1. View ORCID ProfileJohn J. Kelly¹,², 
2. View ORCID ProfileMoe Saee-Marand¹, 
3. View ORCID ProfileNivin N. Nyström¹,², 
4. View ORCID ProfileMelissa M. Evans¹, 
5. View ORCID ProfileYuanxin Chen¹, 
6. Francisco M. Martinez¹, 
7. View ORCID ProfileAmanda M. Hamilton¹ and 
8. View ORCID ProfileJohn A. Ronald¹,²,³,*

 See all authors and affiliationsScience Advances  20 Jan 2021:
Vol. 7, no. 4, eabc3791
DOI: 10.1126/sciadv.abc3791

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Abstract

Imaging reporter genes provides longitudinal information on the biodistribution, growth, and survival of engineered cells in vivo. A translational bottleneck to using reporter genes is the necessity to engineer cells with randomly integrating vectors. Here, we built homology-independent targeted integration (HITI) CRISPR-Cas9 minicircle donors for precise safe harbor-targeted knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency using HITI vectors compared to homology-directed repair vectors. HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significant Oatp1a1-mediated uptake of the clinically approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. Contrast-enhanced MRI improved the conspicuity of both subcutaneous and metastatic Oatp1a1-expressing tumors before they became palpable or even readily visible on precontrast images. Our work demonstrates the first CRISPR-Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multimodal longitudinal in vivo imaging of cells.

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INTRODUCTION

Molecular-genetic imaging with reporter genes permits the in vivo visualization and tracking of engineered cells and thus allows one to track the biodistribution, persistence, viability, and, in some cases, activation state of such cells (1, 2). Several reporter genes currently exist for visualizing engineered cells using preclinical optical fluorescence imaging (FLI) and bioluminescence imaging (BLI) (3–5) as well as those for clinical modalities such as magnetic resonance imaging (MRI), positron emission tomography (PET), and photoacoustic imaging (6–8). These noninvasive cell tracking tools are invaluable for understanding mechanisms of disease progression and the evaluation of treatments in preclinical animal models. Important examples in cancer research include the tracking of therapeutic stem cells (9–11); tracking immune cell migration, cancer progression, and metastasis (12–14); and evaluating tumor response to novel anticancer therapeutics (15, 16). More recently, the use of reporter genes to track therapeutic cells has been translated into the clinic. In this case, cytotoxic T cells were engineered to express a chimeric antigen receptor to target glioma cells, as well as a herpes simplex virus type 1 thymidine kinase (HSV1-TK) dual reporter-suicide gene (that selectively uptakes the PET tracer [¹⁸F]FHBG) to track the localization and viability of the injected therapeutic cells in glioma patients (17, 18).

Although reporter genes have great potential for therapeutic cell tracking, their functionality is best used when the genes are stably integrated into the desired cell’s genome, allowing reporter gene expression throughout the lifetime of the cell and in any subsequent daughter cells. Retroviral vectors, such as those derived from HIV lentiviruses, have generally been used for transgene integration due to their high transfection efficiency, large transgene capacity, and their ability to transduce a variety of dividing and nondividing cell types. However, the low acceptance of using reporter genes for tracking cell-based therapies may, in part, be due to the increased risk of random or quasi-random insertional mutagenesis when transgenes are delivered using viral vectors (19). In previous clinical trials involving children with X-linked severe combined immunodeficiency, a Moloney murine leukemia virus–based γ-retrovirus vector expressing the interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients. However, 5 of the 20 patients also developed leukemia, of which one child died, as a result of insertional mutagenesis and transactivation of proto-oncogenes (20–22). An alternative to viral-based engineering is to use nonviral transposase-based systems such as the Sleeping Beauty or piggyBac transposon systems (23, 24). Transposase systems can integrate large expression cassettes into mammalian cells, but lack specificity, tending to integrate at multiple sites within the genome almost randomly, or with preference for transcriptional start sites and long terminal repeat elements. For future cell-based therapies, it is therefore highly desirable to edit cells with reporter genes in a safe and site-specific manner. The application of such editing tools would allow longitudinal cell tracking to confirm that the cells are performing their intended role and to detect any ectopic growths or misplaced targeting at the earliest time point. This will ultimately give the clinician greater control and confidence in the outcomes of the targeted therapy.

Genomic safe harbors can incorporate exogenous pieces of DNA and permit their predictable function but do not cause alterations to the host genome or pose a risk to the host cell or organism (25). Several studies have successfully used genome editing tools such as zinc finger nucleases (ZFNs) and transcription activator–like effector nucleases (TALENs) to incorporate reporter genes at the adeno-associated virus integration site 1 (AAVS1) safe harbor locus, with no detrimental effects (26–28). Although ZFNs and TALENs have shown great promise as targeted DNA editors, they are time consuming, expensive, and challenging to engineer as unique nuclease sequences must be generated for every genomic target. Alternatively, clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR-Cas9), which was developed by several groups in 2013 (29–32), allows quicker, cheaper, and easier-to-design human genome editing. CRISPR-Cas9 uses short guide RNAs [gRNAs; ~20 base pairs (bp) in length] to direct the Cas9 endonuclease to a specific genomic locus and induce a double-stranded DNA break. Both the Cas9 enzyme and gRNA sequences can be encoded in a single plasmid and, when cotransfected with a donor DNA plasmid, can lead to higher homology-directed repair (HDR) knock-in efficiency than previous editing tools (33). We have previously described the first CRISPR-Cas9 system for AAVS1 integration of donor constructs containing an antibiotic resistance selection gene and both fluorescence (tdTomato) and bioluminescence (Firefly luciferase) reporter genes (34). We were able to confirm the correct and stable integration of donor DNA at the AAVS1 site and functional reporter gene expression in vivo. However, some of the limitations of our study include (i) the low editing efficiency (~3.8%) of human embryonic kidney (HEK)–293T cells; (ii) the use of large CRISPR-Cas9 and donor DNA plasmids that contained bacterial and antibiotic resistance genes, which limit transfection efficiency and would have associated safety concerns for clinical translation; and (iii) the lack of a translationally relevant reporter gene. In this study, we aimed to address these limitations by improving the efficiency and clinical safety of reporter gene integration at the AAVS1 safe harbor site and included a translationally relevant reporter gene.

We posited that the low editing efficiency of our first system was due, in part, to reduced transfection and knock-in efficiency, which is common with larger DNA plasmids, and the use of the HDR repair pathway for integration, which is intrinsically inefficient and not readily accessible to nondividing cells (35). In contrast to HDR-mediated DNA repair, the nonhomologous end joining (NHEJ) pathway is active in both proliferating and nonproliferating cells and is generally considered more efficient than HDR in mammalian cells (36). Recent studies have shown that by designing a CRISPR-Cas9 system that includes the same gRNA cut site in the donor vector as the genomic target site, the NHEJ repair pathway will more efficiently lead to transgene integration in zebrafish (37) and mammalian cells (38, 39). Suzuki et al. (40) refer to this mechanism as homology-independent targeted integration (HITI), which is expected to lead to increased insertion in the forward rather than the reverse direction, as intact gRNA target sequences will be preserved in the latter. Therefore, we postulated that HITI will increase the efficiency of reporter gene integration at the AAVS1 site (Fig. 1A) compared to HDR. To address the problem of size and bacterial/antibiotic resistance genes in plasmids, our group and Suzuki et al. (40) previously designed minicircles (MCs) to express genes of interest (41, 42). First described by Darquet et al. (43), MCs lack the bacterial backbone and antibiotic resistance genes that would otherwise compromise biosafety and clinical translation. In addition, the removal of the prokaryotic backbone also greatly reduces the size of the vector, thus improving transfection efficiency or providing space for the inclusion of other transgenes. To that end, we aimed to improve on our previous work by including a translationally relevant reporter gene in a multimodality imaging HITI MC donor. We determined that the rat organic-anion-transporting polypeptide 1A1 (Oatp1a1) gene was an ideal candidate. Oatp1a1 is a positive contrast MRI reporter gene due to its ability to uptake a clinically approved, liver-specific paramagnetic contrast agent called gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA; Primovist/Eovist) (44). We have previously shown that Oatp1a1 is a sensitive, quantitative, MRI reporter for three-dimensional (3D) cancer cell distribution in vivo (45). The purpose of this study was to develop HITI MC donor vectors for CRISPR-Cas9 editing of cells at the AAVS1 locus with three reporter genes to allow multimodality, longitudinal in vivo monitoring of their fate following transplantation.

RESULTS

CRISPR-Cas9 engineering of multiple human cell types with trimodal reporter gene MCs

In this study, we designed our trimodal reporter gene system in MC constructs to reduce the size and immunogenicity of our donor DNA and to remove antibiotic resistance genes. To compare the efficiency of HDR versus HITI editing at the AAVS1 site, we designed two donor and two Cas9-expressing MCs, as shown in Fig. 1B. The HDR and HITI constructs were engineered to express tdTomato (tdT), firefly luciferase (Fluc2), and rat organic anion transporting polypeptide 1a1 (Oatp1a1) genes under the control of an EF1α promoter and 2A self-cleaving peptide system (Fig. 1B). The HDR and HITI parental plasmids (PPs) initially measured 11.9 and 10.4 kb in size, which were then reduced to 7.9 and 6.4 kb when recombined into MCs, respectively, as confirmed by agarose gel electrophoresis (Fig. 1C). The HDR-MC was flanked by left and right AAVS1 homologous arms either side of the AAVS1 genomic cut site, whereas the HITI donor contained the same CRISPR-Cas9 cut site as the AAVS1 genomic site (fig. S1). In this instance, if the MC DNA inserted in the correct orientation at the AAVS1 site, the CRISPR-Cas9 cut sites would be lost and the trimodal reporter genes would be stably integrated into the genome (fig. S1). The Cas9-expressing MCs were designed to contain the necessary RNA scaffolding and gRNA sequences targeting the AAVS1 site or a scrambled gRNA control, alongside a zsGreen (zsG) fluorescent reporter gene (Fig. 1B). Both the pCas9-AAVS1-MC and pCas9-scrambled-MC constructs measured 12.5 kb in PP form and 8.6 kb in MC form (Fig. 1B).

Our first objective was to determine the correct integration of our donor MCs in three human cell lines: HEK-293T, HeLa, and PC3 cells. All three were cotransfected with the HDR-MC or HITI-MC together with either the Cas9-AAVS1-MC or Cas9-scrambled-MC (as outlined in Fig. 1D) and grown for 48 hours. The cells were then fluorescence-activated cell (FAC) sorted for tdT⁺/zsG⁺ cells to purify cells that were successfully cotransfected, and tdT fluorescence was then tracked every 7 days using flow cytometry (fig. S2, A and B). In two separate experimental groups, the cells were then resorted 14 or 21 days later for tdT⁺/zsG⁻ cells to ensure that the cell populations had not randomly integrated the Cas9-zsG MCs into the genome (fig. S2C). Both PC3 experimental groups were resorted 14 days after the initial sort (and not 21 days later) due to lower transfection rates. However, resorting the cells 14 or 21 days later had a negligible effect on tdT⁺ cell populations across the time points. For almost all cell types, there was a higher percentage of tdT⁺fluorescence cells at end point in the HITI-AAVS1 groups (pink shading, fig. S2C), suggesting better or more stable integration compared to HDR-AAVS1 groups. For the 293T and HeLa cell groups, specifically, the difference was at least two to three times greater for HITI-transfected cells versus HDR. The only exception was the PC3 #1 group, which had a higher incidence of HDR-AAVS1 tdT⁺ cells and was likely a result of poor transfection efficiency of the HITI construct for that group.

MCP integration and BLI analysis

We next performed junctional polymerase chain reaction (PCR) analysis on extracted DNA samples to determine whether the tdT⁺ mixed cell populations (MCPs) had correctly incorporated the trimodal donor MCs into the AAVS1 site in the right orientation (fig. S3A). A correct integration band (1.4 kb) was detected for all HITI-guideAAVS1 (HITIgA)–engineered cells (very low transfection efficiency for PC3 cells may explain why the integration band was weak) as well as a correct integration band (1.3 kb) for HDR-guideAAVS1 (HDRgA) cells for 293T and HeLa MCPs. There were no integration bands for the control naïve cells or cells engineered with scrambled gRNA (HITI/HDRgS). Next, we performed in vitro BLI experiments to determine whether the integrated reporter gene was functioning in the MCPs. Varying numbers of each cell type were imaged with BLI after addition of D-luciferin to visualize FLuc2 expression (fig. S3B). In all cell types, there was a positive correlation between BLI signal and cell number (fig. S3B). There was a consistently higher signal seen in the HITIgA cell populations compared to HDRgA, with approximately three times higher average radiance for the 293T and HeLa HITIgA MCPs and almost six times greater for PC3 HITIgA cells at a concentration of 1 × 10⁵ cells (fig. S3B, right).

HITI is more efficient than HDR in clonal populations

Next, we used clonal cell isolation to determine whether HITI or HDR was more efficient at correctly integrating our large donor MCs at the AAVS1 site. Single-cell tdT⁺ clones were isolated from the 293T and PC3 MCPs into 96-well plates during a third FAC sort (FACS). We decided to use the 293T cells as a proof-of-principle cell line and the PC3 cells as a relevant prostate cancer model cell line; hence, the HeLa cells were not included in studies from this point onward. PCR integration checks were performed on the 293T and PC3 clonal populations to determine the efficiency of HITI- versus HDR-mediated reporter gene integration at the AAVS1 site (Fig. 2, A and B). The number of 293T clonal populations with correct integration was 11.8% (4 of 34) for HDRgA-engineered cells and 36.1% (13 of 36) for HITIgA clones (Fig. 2, A and B). PC3 cells grew fewer colonies but showed zero integration at the AAVS1 site for tdT⁺ HDR-engineered cells (0 of 14), whereas 10.5% (2 of 19) of the HITI-engineered colonies had correct reporter gene integration, indicating that HITI was more efficient in both cell types.

In vitro reporter gene imaging

Next, we expanded single 293T and PC3 HITIgA clonal cells (with correct integration bands) for further in vitro reporter gene functionality testing. First, we confirmed tdT fluorescence for both the 293T-HITI (Fig. 3A) and PC3-HITI (Fig. 3G) clones via fluorescence microscopy. Next, we confirmed a positive correlation between BLI signal and increasing cell numbers for 293T (Fig. 3, B and C; r² = 0.9718) and PC3 (Fig. 3, H and I; r² = 0.9897) cells. BLI signal measured to at least 10 passages showed stable FLuc2 expression over time for both clonal cell lines (Fig. 3, D and J). To test for Oatp1a1 functionality, 293T naïve, 293-HITI, PC3 naïve, and PC3-HITI cells were incubated with or without Gd-EOB-DTPA (6.4 mM) in normal medium for 90 min, washed thoroughly, pelleted, and inserted into an agarose phantom. Inversion recovery MRI was performed at 3 T, and spin-lattice relaxation rate (R₁) maps were generated for 293T (Fig. 3E) and PC3 (Fig. 3K) cell populations. Neither the naïve 293T/PC3 nor untreated 293T-HITI and PC3-HITI cell populations exhibited any change in R₁ rates (Fig. 3, F and L). Only HITI clones expressing Oatp1a1 had significantly increased R₁ rates after Gd-EOB-DTPA incubation, with ~10-fold increase for 293T-HITI cells (7.952 ± 0.87 s⁻¹) compared with naïve, treated controls (0.806 ± 0.038 s⁻¹; n = 3, P < 0.001; Fig. 3F) and ~5-fold increase for PC3-HITI cells (3.426 ± 0.217 s⁻¹) compared with naïve, treated controls (0.6402 ± 0.045 s⁻¹; n = 3, P < 0.001; Fig. 3L).

Oatp1a1 sensitivity

The MR detection limit of Oatp1a1-expressing 293T and PC3 HITI cell clones was investigated by varying the ratio of naïve:HITI cells in MR phantoms and in vivo with subcutaneous PC3-HITI cell injections. In all instances, MRI and BLI were performed on the same day of the experimental setup. For MR phantoms, all cells (except a naïve-no Gd control, “naïve-NG”) were incubated with Gd-EOB-DTPA (5.2 mM, 60 min), thoroughly washed, and then combined so that each 20 × 10⁶ cell sample would contain a defined number of Oatp1a1-expressing HITI cells and naïve cells (expressed as percent-HITI of the total cell population; see Fig. 4 and fig. S4). The cell pellets were then placed into an agarose phantom, and inversion recovery MRI was performed at 3 T. Spin-lattice relaxation rate (R₁) maps were generated for both PC3 (Fig. 4A) and 293T (fig. S4A) cells. Note that the phantoms in Fig. 4A were rearranged in a linear format for figure presentation only and at two different scales—one on a full scale of R₁ values and the second on a saturated scale (maximum 2.0 Hz as the highest concentration) to show visual differences at the lower concentrations. The change in sensitivity with increasing HITI cell numbers can be clearly seen in stacked histograms showing pixel-by-pixel R₁ counts for each PC3 (Fig. 4B) and 293T (fig. S4B) cell pellet. For both cell types, a significant increase in the average R₁ values, compared to naïve controls, was evident when only 10% of the cell pellet contained Oatp1a1-expressing HITI cells (Fig. 4C for PC3 and fig. S4C for 293T).

We next injected various combinations of PC3-naïve and PC3-HITI cells at five sites subcutaneously on the backs of nude mice to analyze Oatp1-expressing cell detection sensitivity in vivo (Fig. 4, D and E, and fig. S5). A total of 3 × 10⁶ cells were injected per site with the following number of PC3-HITI cells: 0 (naïve cell only control), 10⁴, 10⁵, 10⁶, 3 × 10⁶ (PC3-HITI cell only control). Naïve cells were included with HITI cells so that each injection contained a total of 3 × 10⁶ per site. BLI signal intensity increased as PC3-HITI cell numbers increased (representative mouse shown in Fig. 4D), with 10⁶ and 3 × 10⁶ HITI injections showing significant signal increase above naïve background controls (Fig. 4E). Transverse MR images from the same mouse showed positive contrast at both the 10⁶ and 3 × 10⁶ HITI injection sites 5 hours after Gd-EOB-DTPA injection (Fig. 4F). Similar to the BLI data, these sites also exhibited significantly higher contrast-to-noise ratios (CNRs) than naïve controls (Fig. 4F). The 10⁴ and 10⁵ PC3-HITI injections were difficult to visualize on MRI as the phosphate-buffered saline (PBS) of these injections spread out over the ~6 hours after injection and had no discernible positive contrast, so they could not be measured. These data were consistent across all three mice (see fig. S5) and showed that the very minimum number of Oatp1a1-expressing cells we could detect with Gd-EOB-DTPA–based MR contrast was 10⁶ cells in a 50-μl subcutaneous injection volume.

Effect of Gd-EOB-DTPA on HITI-engineered Oatp1a1-expressing cells

Trypan blue exclusion assays and cell counts were performed to determine whether the uptake of Gd-EOB-DTPA into Oatp1a1-expressing 293T and PC3 cells affected cell viability and growth over time. Cells were plated (day 0) and incubated with 5.2 mM Gd-EOB-DTPA contrast agent or saline the next (day 1) for 90 min. There was no significant difference in viability or growth rate between 293T-naïve and 293T-HITI cells or between control (saline) or Gd-EOB-DTPA–treated cell groups (fig. S6A). In contrast, the PC3-HITI clone exhibited a slower growth rate compared to the mixed population of naïve cells (fig. S6B). Exposure to Gd-EOB-DTPA, however, did not affect the viability or growth of either cell type compared to their no-Gd (saline) controls (fig. S6B). These data indicate that Gd-EOB-DTPA uptake had no negative effect on the cells. However, the single PC3-HITI clone we obtained was slower growing compared to the mixed cell naïve population.

PC3-HITI Oatp1a1 tumor models for MRI detection

As a proof of principle that our HITI-engineered cells could show Gd-EOB-DTPA–induced positive MRI contrast in subcutaneous tumors, we injected 293T-naïve and 293T-HITI cells on either flank of a nude mouse (fig. S7). For both cell types, the large masses were visible on precontrast images and showed noticeable positive contrast 20 min after Gd-EOB-DTPA injection. However, 5 hours after contrast, the naïve tumor had returned to precontrast background levels, whereas the HITI tumor had very prominent positive contrast that also showed heterogeneity within the tumor mass (fig. S7). This heterogeneity, in contrast, likely reflects areas of viable (enhancing) and nonviable (non-enhancing) Oatp1-expressing cells within the tumors, as we have reported previously (45).

Moving into a more relevant cancer model, we next injected PC3-naïve and PC3-HITI clonal cells subcutaneously on either flank of nude mice and followed BLI and MRI signal changes over time (Fig. 5). At only 11 days after injection, before the tumors were visible or palpable, clear positive contrast was observed for HITI-engineered cells 5 hours after Gd-EOB-DTPA injection, whereas naïve tumors were undistinguishable (Fig. 5A and fig. S8). The same mice were then imaged at day 46, where the naïve tumor was visible due to pooled Gd-EOB-DTPA at 20 min after contrast injection. In a similar fashion to 5 hours after contrast, only the HITI-engineered cells retained the Gd agent and showed bright, positive contrast (Fig. 5B and fig. S9). Contrast-to-noise measurements, tumor size, and estimated cell numbers for PC3-HITI tumors all increased from days 11 to 46 (Fig. 5, C to E). These data suggest that the Oatp1a1 MRI reporter can detect tumor burden at stages where the tumors are not visible or palpable, and tumor growth can be tracked longitudinally with Gd-EOB-DTPA–enhanced MRI.

Last, PC3-HITI cells were injected via the tail vein into immunocompromised NOD scid gamma (NSG) and nude mice to investigate the ability of Oatp1a1 as a reporter gene for visualizing metastases (Fig. 6 and fig. S10). Using BLI as a guide, we were able to detect metastatic tumors in the head of NSG (Fig. 6) and nude (fig. S10) mice, 84 and 114 days after injection, respectively. Tumors were undistinguishable in precontrast MR images for the NSG mouse. However, 5 hours after Gd-EOB-DTPA injection, a cluster of small, enhanced PC3-HITI tumors were clearly visible (yellow arrows, Fig. 6), which equated to an overall tumor size of 2.05 mm³ and, assuming that a 1-cm³ tumor contains around 1 × 10⁹ cells, contained around 2.1 × 10⁶ cells. Similarly, in the nude mouse model, a larger, more defined tumor showed strong enhancement 5 hours after contrast injection (fig. S10), which measured 31.42 mm³ in size and contained ~3.1 × 10⁷ cells. These data indicate the usefulness of this reporter gene for detecting metastatic burden with contrast-enhanced MRI.

DISCUSSION

As personalized medicine and CRISPR-editing become a reality in the clinic, there is a greater need to (i) improve the efficiency, efficacy, and safety of genetically engineered cell therapies, and (ii) improve our understanding of disease progression and treatment response in preclinical models of disease. Reporter gene-based imaging allows us to track the location, viability, growth, and efficacy of such treatments, and in preclinical models of cancer progression and treatment. In this study, we have developed a non–viral vector–based engineering system for large DNA multimodality reporter gene integration into the AAVS1 safe harbor genomic locus. To improve safety further, we used MCs as the DNA vector of choice, which eliminates bacterial DNA contamination and antibiotic resistance genes. In addition, we showed that using the NHEJ repair pathway with HITI could improve DNA editing efficiency in human cells compared to the more commonly used HDR pathway. Last, building off our previous work (34), we have engineered a trimodality reporter gene construct that contains a clinically relevant MRI reporter, Oatp1a1, in addition to fluorescent and bioluminescent genes, which enabled cell sorting and noninvasive BLI/MRI of engineered cells in a preclinical cancer model.

One of the major limitations of engineering cells with large, multimodality reporter gene DNA plasmids is the reduced efficiency of both transfection and gene editing with increasing construct/insert size (46–48). In addition, the presence of bacterial and antibiotic resistance genes in PPs has the potential to exert immunological responses and raises safety concerns. To circumvent these issues, we designed our study to use MCs, which remove the bacterial backbone from PPs and thus reduce the size of the DNA donor constructs. Using MCs instead of PPs allowed us to remove ~4 kb of unwanted DNA from our HDR construct, with a further reduction of ~1.5 kb for the HITI MC when the homologous arm sequences were replaced with a 20-bp gRNA sequence (saving a total of ~5.5 kb). These large-scale reductions thus provided us with room to upgrade our dual-modality tdTomato and FLuc2 reporter gene construct we previously reported (34) to a trimodality reporter gene construct with the addition of the Oatp1a1 MRI reporter (44, 45). To improve safety and translatability, we also removed the puromycin resistance gene to reduce the MC size by a further 600 bp and used FACS of tdTomato-positive cells to obtain mixed and clonal cell populations instead of antibiotic selection. Our final step for improving safety was to design our system to target a “safe harbor” locus in genomic DNA. Several of these loci have now been reported in the literature (49) and are described as sites where inserted genetic elements can function as intended, without causing alterations that would pose a risk to the host cell or organism (25). For this study, we targeted the AAVS1 site found within the human protein phosphatase 1 regulatory subunit 12C (PP1R12C) gene, as this has been one of the best characterized, to date. No known side effects are associated with disrupting the PP1R12C gene; however, it has been reported that mechanisms such as DNA methylation can silence transgenes targeted to this genomic region (50). Because our studies rely on stable reporter gene expression over time for accurate cell detection and proliferation, we investigated whether reporter gene expression in our AAVS1-engineered 293T-HITI and PC3-HITI cell populations changed over time. We found that BLI signal was stable to at least 10 passages and tdT fluorescence was expressed in both cell lines, indicating consistent transgene expression.

We have shown here that HITI-based CRISPR-Cas9 cell engineering is more efficient than the more commonly used HDR method for integrating large DNA donor constructs. Targeted transgene integration is typically achieved using homologous arms and the HDR pathway; however, this mechanism is highly inefficient and not usually active in nondividing cells (35). Our previous study showed that only 3.8% of selected cells were correctly edited using the HDR mechanism (34). In contrast, the HITI method that uses the NHEJ pathway is active in all stages of the cell cycle and in quiescent cells (38) and thus has been used to improve editing efficiency. Using the method described by Suzuki et al. (40), our engineered 293T and PC3 clonal cell populations did have greater DNA integration at the AAVS1 site compared with HDR (36 and 12% for HITI versus 10.5 and 0% for HDR, respectively). However, the NHEJ repair pathway is error prone and often leads to insertions and deletions (indels) at the DNA junctions. Consequently, this mechanism is often taken advantage of to produce DNA disruptions, gene silencing, and knockouts. These issues would need to be considered if using the HITI method for correctional DNA editing and promoter-less vector integration, because these require specific DNA sequences, either upstream or downstream, to be preserved. In this case, we engineered cells with the only requirements being that the transgene inserts into the AAVS1 site (confirmed with junctional PCR) and that the reporter genes are consistently expressed (confirmed with imaging). Therefore, indels at either the 5′ or 3′ junction would likely have a negligible impact on our experiments.

Although we confirmed correct transgene integration at the AAVS1 site in our study, we cannot rule out integration at other off-target sites in HITI- and HDR-engineered populations. Several 293T and PC3 single-cell clonal populations expressed the tdTomato fluorescence reporter gene but did not show integration bands for the AAVS1 site. Evidence suggests that CRISPR-Cas9 is not 100% accurate and off-target effects have been reported as a common problem associated with CRISPR (51, 52). Thus, it is likely that some MCs integrated into off-target Cas9 cut sites in clones where the correct AAVS1 integration bands were absent or that the MCs inserted into the AAVS1 site in the wrong direction. Although HITI is designed to minimize integration in the wrong orientation, the error-prone NHEJ repair mechanism of blunt-ended DNA breaks could lead to indels at the CRISPR-Cas9 cut site boundaries, which could then disrupt the ability of Cas9 to recognize and recut those sites. Although our preliminary data suggest that stable integration in the wrong direction with HITI was absent in our sorted MCPs, there remains a possibility of indel formation using Cas9 HITI. This could be reduced in future studies by adopting a similar method to that recently reported by Li and colleagues (53), where 5-bp overhangs created by Cas12a could lead to more precisely edited genomes, in a process coined microhomology-dependent TI (MITI) (53). Independently of CRISPR, MCs, like plasmids, can also randomly integrate into the genome of cells, albeit at very low rates. Future work will need to analyze the rate of off-target integrations and possible indel disruptions at the CRISPR-Cas9 cut sites using techniques, such as next-generation sequencing, to determine the full safety profile of HITI at safe harbor loci. To improve targeting specificity, studies have shown that high-fidelity Cas9 enzymes in ribonucleoprotein complexes (RNPs), instead of Cas9 DNA vectors, improve on-target activity while reducing off-target editing (54, 55). In combination with RNPs, AAVs are now commonly used as DNA donors for CRISPR experiments due to their high transduction capabilities in hard-to-transfect cell lines, their low risk of random integration, and reduced immunogenic response. However, AAVs are still limited by their loading capacity of ~4.5 kb, which would be a problem for large, multimodality imaging vectors as presented here, but conceivable for future studies where only one imaging reporter gene is required. With these emerging technologies, it is likely that CRISPR gene editing will become highly specific and thus safer in the near future.

We engineered cells with a multimodality reporter gene construct to enable us to go from single cell, optical imaging methods (FLI) to higher-sensitivity whole-animal planar imaging (BLI) and superior 3D high-resolution tomographic imaging (MRI) in animals. This offers several advantages. First, fluorescently activated cell sorting of tdTomato-expressing cells eliminates the need for an antibiotic resistance selection gene, which constitutes a safety risk and has been associated with structural plasmid instabilities (56). Second, the firefly luciferase gene (FLuc2), in combination with its substrate D-luciferin, allows us to directly visualize engineered cells in vivo using BLI. Inclusion of bioluminescent genes in preclinical cancer models is a relatively inexpensive and valuable tool that also allows one to track cell migration and cell seeding in metastatic cancer models, assess cell viability, and follow cell/tumor growth longitudinally (14). A limitation of BLI is that it is restricted to small animal models of disease. However, it is useful for determining sites of cell arrest/seeding/growth and thus can be used in conjunction with other reporter genes as a guide for determining when and where to perform relatively more expensive, higher-resolution clinical imaging, such as MRI (13). To build off our previous dual FLI-BLI study (34), we decided to include the MRI reporter gene, Oatp1a1, as a translationally relevant and sensitive reporter gene to complete our trimodality construct for HITI-based CRISPR engineering. First described by Patrick et al. (44), Oatp1a1 selectively, but reversibly, uptakes the clinically approved Gd³⁺ contrast agent Gd-EOB-DTPA and thus provides positive contrast in T₁-weighted MR images. The authors concluded, therefore, that Oatp1a1-engineered cells and tumors should be easier to detect than the negative contrast generated by T₂ agents, such as superparamagnetic iron oxide (SPIO) and ferromagnetic agents (44, 57). In addition, engineering cells with integrated Oatp1a1 expression means that MR images can be obtained longitudinally to track cell migration and growth, and signal intensity can be directly correlated with cell viability. Last, we and others have found that Oatp1a1 also enhances the uptake of D-luciferin for BLI (45, 58) and the fluorescent dye indocyanine green (using the human ortholog OATP1B3) for both fluorescent (59) and photoacoustic imaging (60), which gives an added advantage of using Oatp1 for multimodality imaging. Because we, and others, have now shown that the human OATP1B3gene also functions as a useful fluorescent, photoacoustic, and MRI reporter gene, in vivo (59, 60), future studies will focus on exchanging Oatp1a1 for the more translationally favorable OATP1B3 ortholog.

In this study, we set out to determine the smallest number of Oatp1a1-expressing HITI-engineered cells that we could detect using MRI phantoms and in vivo injections (with BLI as a guide). Our in vitro MRI phantom experiments showed significant increases in R₁ rates when 10% of the cell pellet, and thus only 10% of each voxel, contained Oatp1a1-expressing cells. For in vivo sensitivity measurements, we were able to reliably detect 1 million cells in a 50-μl volume. The cell and contrast injections and imaging were all performed on the same day so that the cell numbers would not vary with migration, cell death, or proliferation. Similar experiments have been performed for PET reporter genes. However, there are some issues with this experimental setup as we prepared cells in PBS rather than within a matrix such as Matrigel so as not to impede diffusion of the contrast agent. These were merely subcutaneous injections of a known number of cells in a known volume of PBS, not tumors. Without time to develop blood vessels, as you would with tumors, it is likely that some of the contrast agent could not diffuse into the injection site fully. This may explain why the contrast enhancement appears as a ring around the cell injection sites. We also believe that this problem likely contributed to the lack of signal enhancement in the 10⁴ and 10⁵ injection sites, which without a matrix to hold them in place also had the problem of cell spreading over a larger surface area. In summary, there are no easy ways to measure the sensitivity of exact cell numbers in vivo, especially with a reporter gene system that relies on an injected substrate; however, the combination of in vitro and in vivo experiments give us a better idea of the minimum number of cell numbers we can confidently detect.

When expressing reporter genes in cells, it is important to know whether the protein and/or its substrates affect the normal function or viability of the cell. In our study, we noticed that the Oatp1a1-expressing PC3-HITI cells grew slower compared to the mixed cell naïve population in vitro and in vivo, whereas their 293T-HITI counterpart showed no difference compared to naïve cells. Although unfortunate for comparative purposes, we believe that the slow growth rate of the PC3-HITI cells was simply due to heterogeneity in clonal populations—especially when comparing to mixed naïve populations—and not due to expression of Oatp1a1. Our data also support this because the 293T-HITI cells were not affected. In addition, we and others have already shown that there is no impact of Oatp1a1 on cell growth in several different cell types (44, 45). Ideally, several correctly engineered clones would be combined for biological and imaging experiments. We were limited in this case by obtaining just one usable PC3-HITI clone. However, the clone still grew primary and metastatic tumors, which we could sensitively detect with BLI and MRI. In addition, the uptake of Gd-EOB-DTPA did not have any detrimental impact on cell viability or growth, indicating that Oatp1 is a viable option for reporter gene imaging and cell tracking.

The improved safety profile and expression of multimodal reporter genes proposed here could have several uses in cell engineering, or at least help answer several concerns with in vivo cell therapies. For example, the U.S. Food and Drug Administration have listed potential safety concerns related to unproven stem cell therapies (61), including (i) the ability of cells to move from placement sites and change into inappropriate cell types or multiply, (ii) failure of cells to work as expected, and (iii) the growth of tumors. In addition, the long-term safety profiles of cells engineered with randomly integrating viruses still require further investigation and optimization. These are concerns that could be addressed by targeting nonviral DNA vectors, such as MCs, to specific safe harbor loci, such as AAVS1, and reducing the use of integrating viruses. Incorporating reporter genes for clinical grade imaging will also help improve patient safety by allowing one to track cellular therapies in vivo [such as for stem cells or cancer-homing theranostic cells (62)]. Clinicians could then determine whether the therapeutic cells are localizing to the correct anatomical feature, such as a solid tumor (18), or to determine their persistence and viability for short- and long-term treatment strategies. Future work will focus on evaluating our system in stem cells and other clinically relevant cell types. Translation will also need to consider building donor vectors that lack optical reporter genes and use other selection methods (e.g., magnetic sorting). It is easily feasible to switch out genes from our trimodality construct for other imaging purposes, such as replacing FLuc2 with a PET reporter gene for dual PET-MR imaging. Suicide switch genes could also be incorporated to further improve safety by killing the engineered cells in cases where they become oncogenic (63), for example. These tools not only are useful for clinical cell-based therapies but also are extremely useful in preclinical studies for investigating cancer progression/aggression, metastatic burden, and treatment strategies. Avoiding the use of random-integrating viruses and targeted editing should also help reduce off-target effects of gene editing that may alter the normal characteristics of the cell type being studied.

CONCLUSION

Our work demonstrates the first CRISPR-Cas9 HITI MC system for safe harbor integration of a large donor construct encoding three reporter genes for multimodal longitudinal imaging of cells in vivo. We have shown that inclusion of the translationally relevant MR reporter gene, Oatp1a1, can enable localization and tracking of small primary and metastatic tumors that are not readily detectable visually or in precontrast MR images. This work lays the foundation for an effective and safer nonviral genome editing tool for noninvasive reporter gene tracking of multiple cell types in vivo.

MATERIALS AND METHODS

Constructs

Construct designs are shown in Fig. 1B. The pCas9-AAVS1guideRNA-zsG-MC (Cas9-AAVS1-MC) and pCas9-scrambledRNA-zsG-MC (Cas9-scrambled-MC) PPs originated from pCas-Guide-AAVS1 and pCas-Guide-Scrambled plasmids purchased from Origene (MD, USA). The Cas9 enzyme and gRNA sequences were cloned between attB and attP recombination sites in an MC bacterial backbone containing a ZsGreen (zsG) fluorescence reporter driven by the elongation factor 1-α promoter (hEF1α). The AAVS1-HDR-tdT-Fluc2-Oatp1a1-MC (HDR-MC) construct was derived from an HDR vector lacking the Oatp1a1 gene as we described previously (34). This plasmid is driven by the hEF1α promoter and expresses tdTomato (tdT), firefly luciferase (Fluc2), and organic anion transporting polypeptide 1a1 (Oatp1a1) using a self-cleaving 2a peptide system. For improved expression, the plasmids also contain the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) followed by the human growth hormone polyadenylation signal (hGH polyA). The HDR plasmid contains the left and right homologous arms (RHA: 527 bp, LHA: 481 bp) that are complementary to the region flanking the AAVS1 cut site; the homologous arms were obtained from the pAAVS1-puroDNR plasmid from Origene (MA, USA). The Oatp1a1 gene was added through PCR amplification from a previously made vector we constructed using PGK_Straw_E2A_Oatp1a1 (a gift from K. Brindle’s laboratory; University of Cambridge). Using the HDR-MC PP as the template, we generated the pAAVS1-HITI-tdT-Fluc2-Oatp1a1-MC (HITI-MC) PP using the In-Fusion Cloning Kit from Clontech (Takara Bio, CA, USA). Using restriction enzyme digestion, we extracted the bacterial backbone and MC recombination sites and then extracted the three reporter genes (without the homologous arms)—tdT, Fluc2, and Oatp1a1—from the HDR-MC construct using PCR. However, for HITI functionality, we designed our primers to also include a 23-bp extension (5′-GTTAATGTGGCTCTGGTTCTGGG-3′) downstream of the polyA sequence, which incorporates the same cut site and protospacer adjacent motif (PAM) sequence for our AAVS1 gRNA, which allows Cas9 cutting of both the MC and genomic DNA.

MC production

ZYCY10P3S2T Escherichia coli (System Biosciences, Palo Alto, CA, USA) were transformed with the original PPs of all four constructs (HDR-MC or HITI-MC, Cas9-AAVS1-MC, and Cas9-scrambled-MC), and viable colonies were selected using kanamycin plates. Colonies were picked 24 hours after transformation and grown in 6 ml of lysogeny broth (LB) with kanamycin for 6 hours at 37°C, followed by growth in terrific broth (TB) for 12 hours at 37°C. To induce expression of the phiC31 integrase for MC production via attB and attPrecombination, 100 ml of LB broth together with 100 μl of 20% arabinose induction solution (System Biosciences, Palo Alto, CA, USA) and 4 ml of 1 N NaOH was added to the culture and grown for 5.5 hours at 30°C. An endotoxin-free maxi kit (Qiagen, Valencia, CA, USA) was used to purify both PP and MC. Following purification of the MC products, PP contamination was removed using the Plasmid Safe ATP-dependent DNase Kit (Epicentre, WI, USA), and the products were cleaned and concentrated using the Clean & Concentrator-25 Kit (Zymo Research, CA, USA).

Cell culture and transfection

HEK-293T cells and human adenocarcinoma HeLa cells (both from the American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (Wisent Bioproducts, Québec, Canada) supplemented with 10% fetal bovine serum (FBS; Wisent Bioproducts, Québec, Canada) and 1× antibiotic-antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). Human grade 4 adenocarcinoma PC3 cells were a gift from H. Leong (Western University, ON, Canada) and were grown in RPMI (Wisent Bioproducts, Québec, Canada) supplemented with 5% FBS and 1× antibiotic-antimycotic. Cells were transfected with the linear polyethylenimine transfection agent jetPEI (Polyplus-transfection, Illkirch, France), according to the manufacturer’s instructions. Briefly, cells were grown in six-well plates until 80 to 90% confluency and cotransfected with 1 μg each of Cas9-AAVS1-MC or Cas9-Scrambled-MC together with 1 μg of the donor MC constructs: HDR-MC or HITI-MC, for a total DNA mass of 2 μg. The DNA was prepared in 150 mM NaCl and complexed with 4 μl of jetPEI reagent per well.

FACS and flow analysis

All FACS and flow cytometry was performed at the London Regional Flow Cytometry Facility (Robarts Research Institute, London, Canada). Forty-eight hours after transfection, the population of cells displaying both red (tdTomato) and green (zsGreen) fluorescence were sorted using a BD FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA). At selected time points following FACS, the cells were analyzed for tdTomato fluorescence using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA). Either 14 or 21 days after the initial sort, the cells were again sorted on the FACSAria III to purify tdTomato-positive cells only (referred to as the pooled population). In this regard, our protocol aimed to sort cells that had incorporated the MC inserts (based on tdTomato fluorescence) into the genome and excluded any cells that had randomly integrated Cas9 MC DNA (zsGreen). At the same time as the second (tdTomato) sort, individual cells were plated into wells of a 96-well plate to enable single-cell colonies to be grown and expanded (referred to as clonal cell populations).

Genomic DNA extractions and AAVS1 integration analysis

Extraction of genomic DNA from the pooled population of cells was performed using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. DNA quality and concentrations were measured on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Extraction of genomic DNA from clonal populations was performed as we described previously (34). Briefly, cell pellets were resuspended in a QuickExtract DNA extraction solution (Lucigen, Middleton, WI, USA), incubated at 65°C for 10 min, vortexed, and incubated at 98°C for 5 min. The DNA was then directly used for PCR or stored at −20°C. To check for integration at the AAVS1 site, two primers were designed to amplify the 3′ junction between the donor cassette and the AAVS1 site outside of the homologous arm region. The forward primer was uniquely complementary to the polyA tail in the MC cassette (5′-CCTGGAAGTTGCCACTCCAG-3′) and the reverse primer to the AAVS1 site (5′-AAGGCAGCCTGGTAGACAGG-3′). A 1.3-kb PCR product was produced if the MC-HDR was correctly integrated at the AAVS1 site, and a 1.7-kb PCR product was produced if MC-HITI was correctly integrated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were designed as DNA loading controls and to confirm successful DNA extractions: forward 5′-TTGCCCTCAACGACCACTTT-3′ and reverse 5′-GTCCCTCCCCAGCAAGAATG-3′ and yielded a PCR product of 502 bp. Agarose gel electrophoresis with 1% agarose gels and RedSafe (FroggaBio, ON, Canada) was used to separate and visualize PCR products.

In vitro fluorescence and BLI

The pooled and clonal cell populations were evaluated for tdTomato fluorescence expression on an EVOS FL auto 2 microscope (Thermo Fisher Scientific, Waltham, MA, USA). For BLI experiments, varying cell numbers were plated in triplicate into black walled 96-well plates. D-Luciferin (0.1 mg/ml; PerkinElmer, Waltham, MA, USA) was added to each well, and images were rapidly collected on the IVIS Lumina XRMS In Vivo Imaging System (PerkinElmer) equipped with a cooled charge-coupled device (CCD) camera. Average radiance values in photons s⁻¹ cm⁻² sr⁻¹ were measured from regions of interest drawn around each well using LivingImage software (PerkinElmer).

In vitro MRI

Naïve and Oatp1a1-expressing cell clones were seeded in 15-cm tissue culture dishes and grown to confluency. Cells were incubated with medium containing 5.2 mM Gd-EOB-DTPA or with medium containing an equivalent volume of PBS for 90 min at 37°C and 5% CO₂. Cells were then washed three times with PBS, trypsinized, and pelleted (20 × 10⁶ cells per pellet) in 0.2-ml Eppendorf tubes. For sensitivity experiments, various numbers of naïve:HITI cells were combined in Eppendorf tubes, mixed well, and then pelleted. The tubes were placed into a 1% agarose phantom mold, and MRI was performed on a 3-T GE clinical MRI scanner with an eight-channel head RF coil (General Electric Healthcare Discovery MR750 3.0 T, Milwaukee, WI, USA). A fast spin echo inversion recovery (FSE-IR) pulse sequence was used with the following parameters: matrix size, 256 × 256; repetition time (TR), 5000 ms; echo time (TE), 16.3 ms; echo train length (ETL), 4; number of excitations (NEX), 1; receiver bandwidth (rBW), 25 kHz; inversion times (TIs), 25, 50, 100, 200, 350, 500, 750, 1000, 1500, 2000, 2500, and 3000 ms; in-plane resolution, 0.27 mm × 0.27 mm; slice thickness, 2.0 mm; scan time, 5 min and 25 s per TI. Spin-lattice relaxation rates (R₁) were determined by nonlinear least-squares fitting (MATLAB, MathWorks, Natick, MA, USA) of the following equation to the signal intensity across the series of TIs on a pixel by pixel basisS=∣Mss−(Mss−Mi)·e−TIT1/∣

Here, S, M_ss, and M_i represent the acquired signal, the longitudinal magnetization in steady-state equilibrium, and the initial longitudinal magnetization acquired after the inversion pulse, respectively. T₁ is the spin-lattice relaxation time such that R1=T−11 and TI is the inversion time.

Animal models

All animal protocols were approved by the University Council on Animal Care at the University of Western Ontario (protocol #2015-058) and follow the Canadian Council on Animal Care (CCAC) and Ontario Ministry of Agricultural, Food and Rural Affairs (OMAFRA) guidelines. Crl:NU-Foxn1^nu (nude) male mice (Charles River Laboratories, Wilmington, MA, USA; N = 3 to 5) aged 6 to 8 weeks were used for subcutaneous and metastatic tumor model injections, and NOD.Cg-Prkdc^scid Il2rg^tm1WjI/SzJ (NSG) immunodeficient male mice (obtained from the Humanized Mouse and Xenotransplantation Facility at the Robarts Research Institute, University of Western Ontario, London, Canada; N = 3) were used for experimental metastasis models (intravenous cell injections).

In vivo BLI

BLI was performed on the same IVIS Lumina XRMS system described for in vitro imaging. Mice were anesthetized with 2% isoflurane in 100% oxygen using a nose cone attached to an activated carbon charcoal filter for passive scavenging and kept warm on a heated stage. Anesthetized mice received a 100-μl intraperitoneal injection of D-luciferin (30 mg/ml), and BLI images were acquired with automatic exposure times until the peak BLI signal was obtained (up to 40 min). Regions of interest were manually drawn using LivingImage software to measure average radiance (photons s⁻¹ cm⁻² sr⁻¹). The peak average radiance was used for quantification for each mouse.

In vivo MRI and quantification

All mouse MRI scans were performed with a custom-built gradient insert and a 3.5-cm-diameter birdcage RF coil (Morris Instruments, Ottawa, ON, Canada), as we described previously (45). Mice were kept anesthetized during the scan with 2% isoflurane administered via a nose cone attached to the coil. T₁-weighted images were acquired using a 3D spoiled gradient recalled acquisition in steady-state pulse sequence using the following parameters: field of view, 50 mm; TR, 14.7 ms; TE, 3.3 ms; rBW, 31.25 MHz; matrix size, 250 × 250; flip angle, 60°; NEX, 3; 200-μm isotropic voxels; scan time, approximately 15 min per mouse. Precontrast images were acquired followed by administration of Gd-EOB-DTPA (1.67 mmol/kg) (Primovist; Bayer, Mississauga, ON, Canada) via the tail vein. Mice were then reimaged 20 min later for immediate postcontrast images, which provide positive contrast to many tissues, including the naïve tumors, as a result of Gd-EOB-DTPA pooling, and/or 5 hours later for Oatp1a1-specific uptake. This time point was determined to allow enough time for Gd-EOB-DTPA to be cleared, yet still provided strong positive contrast in Oatp1a1-expressing cells (44, 45). CNR and tumor size measurements were calculated from MR images using ITK-snap open source software (www.itksnap.org) (64). Tumors were manually segmented in three dimensions by tracing the tumor or control tissue (hind leg muscle) with polygon and paintbrush tools and pixel intensity recorded in every slice. The CNR of tumors was calculated by taking the signal intensity of the difference between tumor regions and muscle tissue divided by the SD of background signal(CNR=attenuationtumor−attenuationmuscle Std.Dev.background)

The number of cells in the tumors was estimated from the assumption that a tumor reaching a size of 1 cm³is estimated to contain around 1 × 10⁹ cells.

In vivo Oatp1a1-induced Gd-EOB-DTPA uptake MRI and BLI sensitivity

To evaluate the cellular detection sensitivity of Oatp1a1-expressing cells with Gd-EOB-DTPA–enhanced MRI, nude mice were injected with 50 μl of cell suspensions in PBS containing 3 × 10⁶ total cells per injection at the following ratios: 3 × 10⁶ naïve cells alone; 10⁴ PC3-HITI + 2.99 × 10⁶ naïve cells; 10⁵ PC3-HITI + 2.9 × 10⁶naïve cells; 10⁶ PC3-HITI + 2 × 10⁶ naïve cells; and 3 × 10⁶ PC3-HITI cells alone, subcutaneously in five locations on the back/flank region. Immediately after cell injections, Gd-EOB-DTPA (1.67 mmol/kg) was injected into the tail vein, and mice were imaged on a 3-T clinical grade MR scanner 5 hours later. This time point allows clearance of Gd-EOB-DTPA from the body yet provides sufficient time for the agent to penetrate the subcutaneous injections sites and accumulate in Oatp1a1-expressing cells. After MRI, mice were moved to the IVIS scanner and injected with 100 μl of D-luciferin (30 mg/ml) intraperitoneally and BLI was performed, as described earlier.

293T and PC3 tumor models

293T or PC3 naïve and HITI-engineered cells were injected subcutaneously (2.5 × 10⁶ 293Ts and 1 × 10⁶PC3s) on the left and right flanks of nude mice, respectively (293T, N = 2; PC3, N = 5). For experimental metastasis studies, 5 × 10⁵ PC3 naïve or HITI-engineered cells were injected into the tail veins of NSG or nude mice (N = 3). Tumor growth was tracked on a weekly basis with BLI, as described above. MRI was performed on mice at various time points, as indicated in the results section. First, a precontrast scan was performed on all mice, followed immediately with injection of the Gd-EOB-DTPA contrast agent into the tail vein (1.67 mmol/kg). For some experiments, the mice were rescanned 15 to 20 min after contrast injection to show tumor and whole-body distribution of Gd-EOB-DTPA. In all instances, MRI scans were performed ~5 hours after contrast injection because Oatp1a1-expressing cells still retain Gd-EOB-DTPA and show strong positive contrast at this time point. This also allows enough time for washout of Gd-EOB-DTPA in most tissues and organs (except for the gastrointestinal tract and bladder where cleared Gd-EOB-DTPA accumulates before being excreted) (44).

Statistical analysis

Statistical analysis was performed with GraphPad Prism version 7 (GraphPad Software Inc., CA, USA; www.graphpad.com) software. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used for in vitro and in vivo BLI and CNR data analysis. An unpaired one-tailed t test with Welch’s correction was used to analyze the increase in CNR/cell numbers for PC3-HITI day 11 versus day 46 tumors.

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/7/4/eabc3791/DC1

View/request a protocol for this paper from Bio-protocol.https://creativecommons.org/licenses/by-nc/4.0/

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

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Acknowledgments: We thank K. Brindle (University of Cambridge) for providing the Oatp1a1 plasmid, Y. Xia for help with paper revisions, K. Chadwick for FACS and flow cytometry help, and D. Reese for MRI troubleshooting. Funding: This work was funded by a Canadian Institutes of Health Research (CIHR) project grant (grant #377071, J.A.R.), a Natural Sciences and Engineering Research Council (NSERC) discovery grant (grant #RGPIN-2016-05420, J.A.R.), and a University of Western Ontario Strategic Support for CIHR Success Seed grant (J.A.R.). Author contributions: J.A.R. designed the project. J.A.R., J.J.K., and M.S.-M. designed the experiments. J.J.K. directed the study and, with M.S.-M., carried out most of the experiments. N.N.N. developed the methods for Oatp1a1 MRI, and F.M.M. developed the MATLAB scripts for processing MRI phantom data. Y.C. helped perform MRI. M.M.E. processed and analyzed MRI data. A.M.H. helped develop the PPs. J.J.K. wrote the manuscript with help from M.S.-M., which J.A.R. reviewed and edited. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

• Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

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Safe harbor-targeted CRISPR-Cas9 homology-independent targeted integration for multimodality reporter gene-based cell tracking

1. View ORCID ProfileJohn J. Kelly¹,², 
2. View ORCID ProfileMoe Saee-Marand¹, 
3. View ORCID ProfileNivin N. Nyström¹,², 
4. View ORCID ProfileMelissa M. Evans¹, 
5. View ORCID ProfileYuanxin Chen¹, 
6. Francisco M. Martinez¹, 
7. View ORCID ProfileAmanda M. Hamilton¹ and 
8. View ORCID ProfileJohn A. Ronald¹,²,³,*

 See all authors and affiliationsScience Advances  20 Jan 2021:
Vol. 7, no. 4, eabc3791
DOI: 10.1126/sciadv.abc3791

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Abstract

Imaging reporter genes provides longitudinal information on the biodistribution, growth, and survival of engineered cells in vivo. A translational bottleneck to using reporter genes is the necessity to engineer cells with randomly integrating vectors. Here, we built homology-independent targeted integration (HITI) CRISPR-Cas9 minicircle donors for precise safe harbor-targeted knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency using HITI vectors compared to homology-directed repair vectors. HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significant Oatp1a1-mediated uptake of the clinically approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. Contrast-enhanced MRI improved the conspicuity of both subcutaneous and metastatic Oatp1a1-expressing tumors before they became palpable or even readily visible on precontrast images. Our work demonstrates the first CRISPR-Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multimodal longitudinal in vivo imaging of cells.

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INTRODUCTION

Molecular-genetic imaging with reporter genes permits the in vivo visualization and tracking of engineered cells and thus allows one to track the biodistribution, persistence, viability, and, in some cases, activation state of such cells (1, 2). Several reporter genes currently exist for visualizing engineered cells using preclinical optical fluorescence imaging (FLI) and bioluminescence imaging (BLI) (3–5) as well as those for clinical modalities such as magnetic resonance imaging (MRI), positron emission tomography (PET), and photoacoustic imaging (6–8). These noninvasive cell tracking tools are invaluable for understanding mechanisms of disease progression and the evaluation of treatments in preclinical animal models. Important examples in cancer research include the tracking of therapeutic stem cells (9–11); tracking immune cell migration, cancer progression, and metastasis (12–14); and evaluating tumor response to novel anticancer therapeutics (15, 16). More recently, the use of reporter genes to track therapeutic cells has been translated into the clinic. In this case, cytotoxic T cells were engineered to express a chimeric antigen receptor to target glioma cells, as well as a herpes simplex virus type 1 thymidine kinase (HSV1-TK) dual reporter-suicide gene (that selectively uptakes the PET tracer [¹⁸F]FHBG) to track the localization and viability of the injected therapeutic cells in glioma patients (17, 18).

Although reporter genes have great potential for therapeutic cell tracking, their functionality is best used when the genes are stably integrated into the desired cell’s genome, allowing reporter gene expression throughout the lifetime of the cell and in any subsequent daughter cells. Retroviral vectors, such as those derived from HIV lentiviruses, have generally been used for transgene integration due to their high transfection efficiency, large transgene capacity, and their ability to transduce a variety of dividing and nondividing cell types. However, the low acceptance of using reporter genes for tracking cell-based therapies may, in part, be due to the increased risk of random or quasi-random insertional mutagenesis when transgenes are delivered using viral vectors (19). In previous clinical trials involving children with X-linked severe combined immunodeficiency, a Moloney murine leukemia virus–based γ-retrovirus vector expressing the interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients. However, 5 of the 20 patients also developed leukemia, of which one child died, as a result of insertional mutagenesis and transactivation of proto-oncogenes (20–22). An alternative to viral-based engineering is to use nonviral transposase-based systems such as the Sleeping Beauty or piggyBac transposon systems (23, 24). Transposase systems can integrate large expression cassettes into mammalian cells, but lack specificity, tending to integrate at multiple sites within the genome almost randomly, or with preference for transcriptional start sites and long terminal repeat elements. For future cell-based therapies, it is therefore highly desirable to edit cells with reporter genes in a safe and site-specific manner. The application of such editing tools would allow longitudinal cell tracking to confirm that the cells are performing their intended role and to detect any ectopic growths or misplaced targeting at the earliest time point. This will ultimately give the clinician greater control and confidence in the outcomes of the targeted therapy.

Genomic safe harbors can incorporate exogenous pieces of DNA and permit their predictable function but do not cause alterations to the host genome or pose a risk to the host cell or organism (25). Several studies have successfully used genome editing tools such as zinc finger nucleases (ZFNs) and transcription activator–like effector nucleases (TALENs) to incorporate reporter genes at the adeno-associated virus integration site 1 (AAVS1) safe harbor locus, with no detrimental effects (26–28). Although ZFNs and TALENs have shown great promise as targeted DNA editors, they are time consuming, expensive, and challenging to engineer as unique nuclease sequences must be generated for every genomic target. Alternatively, clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR-Cas9), which was developed by several groups in 2013 (29–32), allows quicker, cheaper, and easier-to-design human genome editing. CRISPR-Cas9 uses short guide RNAs [gRNAs; ~20 base pairs (bp) in length] to direct the Cas9 endonuclease to a specific genomic locus and induce a double-stranded DNA break. Both the Cas9 enzyme and gRNA sequences can be encoded in a single plasmid and, when cotransfected with a donor DNA plasmid, can lead to higher homology-directed repair (HDR) knock-in efficiency than previous editing tools (33). We have previously described the first CRISPR-Cas9 system for AAVS1 integration of donor constructs containing an antibiotic resistance selection gene and both fluorescence (tdTomato) and bioluminescence (Firefly luciferase) reporter genes (34). We were able to confirm the correct and stable integration of donor DNA at the AAVS1 site and functional reporter gene expression in vivo. However, some of the limitations of our study include (i) the low editing efficiency (~3.8%) of human embryonic kidney (HEK)–293T cells; (ii) the use of large CRISPR-Cas9 and donor DNA plasmids that contained bacterial and antibiotic resistance genes, which limit transfection efficiency and would have associated safety concerns for clinical translation; and (iii) the lack of a translationally relevant reporter gene. In this study, we aimed to address these limitations by improving the efficiency and clinical safety of reporter gene integration at the AAVS1 safe harbor site and included a translationally relevant reporter gene.

We posited that the low editing efficiency of our first system was due, in part, to reduced transfection and knock-in efficiency, which is common with larger DNA plasmids, and the use of the HDR repair pathway for integration, which is intrinsically inefficient and not readily accessible to nondividing cells (35). In contrast to HDR-mediated DNA repair, the nonhomologous end joining (NHEJ) pathway is active in both proliferating and nonproliferating cells and is generally considered more efficient than HDR in mammalian cells (36). Recent studies have shown that by designing a CRISPR-Cas9 system that includes the same gRNA cut site in the donor vector as the genomic target site, the NHEJ repair pathway will more efficiently lead to transgene integration in zebrafish (37) and mammalian cells (38, 39). Suzuki et al. (40) refer to this mechanism as homology-independent targeted integration (HITI), which is expected to lead to increased insertion in the forward rather than the reverse direction, as intact gRNA target sequences will be preserved in the latter. Therefore, we postulated that HITI will increase the efficiency of reporter gene integration at the AAVS1 site (Fig. 1A) compared to HDR. To address the problem of size and bacterial/antibiotic resistance genes in plasmids, our group and Suzuki et al. (40) previously designed minicircles (MCs) to express genes of interest (41, 42). First described by Darquet et al. (43), MCs lack the bacterial backbone and antibiotic resistance genes that would otherwise compromise biosafety and clinical translation. In addition, the removal of the prokaryotic backbone also greatly reduces the size of the vector, thus improving transfection efficiency or providing space for the inclusion of other transgenes. To that end, we aimed to improve on our previous work by including a translationally relevant reporter gene in a multimodality imaging HITI MC donor. We determined that the rat organic-anion-transporting polypeptide 1A1 (Oatp1a1) gene was an ideal candidate. Oatp1a1 is a positive contrast MRI reporter gene due to its ability to uptake a clinically approved, liver-specific paramagnetic contrast agent called gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA; Primovist/Eovist) (44). We have previously shown that Oatp1a1 is a sensitive, quantitative, MRI reporter for three-dimensional (3D) cancer cell distribution in vivo (45). The purpose of this study was to develop HITI MC donor vectors for CRISPR-Cas9 editing of cells at the AAVS1 locus with three reporter genes to allow multimodality, longitudinal in vivo monitoring of their fate following transplantation.

RESULTS

CRISPR-Cas9 engineering of multiple human cell types with trimodal reporter gene MCs

In this study, we designed our trimodal reporter gene system in MC constructs to reduce the size and immunogenicity of our donor DNA and to remove antibiotic resistance genes. To compare the efficiency of HDR versus HITI editing at the AAVS1 site, we designed two donor and two Cas9-expressing MCs, as shown in Fig. 1B. The HDR and HITI constructs were engineered to express tdTomato (tdT), firefly luciferase (Fluc2), and rat organic anion transporting polypeptide 1a1 (Oatp1a1) genes under the control of an EF1α promoter and 2A self-cleaving peptide system (Fig. 1B). The HDR and HITI parental plasmids (PPs) initially measured 11.9 and 10.4 kb in size, which were then reduced to 7.9 and 6.4 kb when recombined into MCs, respectively, as confirmed by agarose gel electrophoresis (Fig. 1C). The HDR-MC was flanked by left and right AAVS1 homologous arms either side of the AAVS1 genomic cut site, whereas the HITI donor contained the same CRISPR-Cas9 cut site as the AAVS1 genomic site (fig. S1). In this instance, if the MC DNA inserted in the correct orientation at the AAVS1 site, the CRISPR-Cas9 cut sites would be lost and the trimodal reporter genes would be stably integrated into the genome (fig. S1). The Cas9-expressing MCs were designed to contain the necessary RNA scaffolding and gRNA sequences targeting the AAVS1 site or a scrambled gRNA control, alongside a zsGreen (zsG) fluorescent reporter gene (Fig. 1B). Both the pCas9-AAVS1-MC and pCas9-scrambled-MC constructs measured 12.5 kb in PP form and 8.6 kb in MC form (Fig. 1B).

Our first objective was to determine the correct integration of our donor MCs in three human cell lines: HEK-293T, HeLa, and PC3 cells. All three were cotransfected with the HDR-MC or HITI-MC together with either the Cas9-AAVS1-MC or Cas9-scrambled-MC (as outlined in Fig. 1D) and grown for 48 hours. The cells were then fluorescence-activated cell (FAC) sorted for tdT⁺/zsG⁺ cells to purify cells that were successfully cotransfected, and tdT fluorescence was then tracked every 7 days using flow cytometry (fig. S2, A and B). In two separate experimental groups, the cells were then resorted 14 or 21 days later for tdT⁺/zsG⁻ cells to ensure that the cell populations had not randomly integrated the Cas9-zsG MCs into the genome (fig. S2C). Both PC3 experimental groups were resorted 14 days after the initial sort (and not 21 days later) due to lower transfection rates. However, resorting the cells 14 or 21 days later had a negligible effect on tdT⁺ cell populations across the time points. For almost all cell types, there was a higher percentage of tdT⁺fluorescence cells at end point in the HITI-AAVS1 groups (pink shading, fig. S2C), suggesting better or more stable integration compared to HDR-AAVS1 groups. For the 293T and HeLa cell groups, specifically, the difference was at least two to three times greater for HITI-transfected cells versus HDR. The only exception was the PC3 #1 group, which had a higher incidence of HDR-AAVS1 tdT⁺ cells and was likely a result of poor transfection efficiency of the HITI construct for that group.

MCP integration and BLI analysis

We next performed junctional polymerase chain reaction (PCR) analysis on extracted DNA samples to determine whether the tdT⁺ mixed cell populations (MCPs) had correctly incorporated the trimodal donor MCs into the AAVS1 site in the right orientation (fig. S3A). A correct integration band (1.4 kb) was detected for all HITI-guideAAVS1 (HITIgA)–engineered cells (very low transfection efficiency for PC3 cells may explain why the integration band was weak) as well as a correct integration band (1.3 kb) for HDR-guideAAVS1 (HDRgA) cells for 293T and HeLa MCPs. There were no integration bands for the control naïve cells or cells engineered with scrambled gRNA (HITI/HDRgS). Next, we performed in vitro BLI experiments to determine whether the integrated reporter gene was functioning in the MCPs. Varying numbers of each cell type were imaged with BLI after addition of D-luciferin to visualize FLuc2 expression (fig. S3B). In all cell types, there was a positive correlation between BLI signal and cell number (fig. S3B). There was a consistently higher signal seen in the HITIgA cell populations compared to HDRgA, with approximately three times higher average radiance for the 293T and HeLa HITIgA MCPs and almost six times greater for PC3 HITIgA cells at a concentration of 1 × 10⁵ cells (fig. S3B, right).

HITI is more efficient than HDR in clonal populations

Next, we used clonal cell isolation to determine whether HITI or HDR was more efficient at correctly integrating our large donor MCs at the AAVS1 site. Single-cell tdT⁺ clones were isolated from the 293T and PC3 MCPs into 96-well plates during a third FAC sort (FACS). We decided to use the 293T cells as a proof-of-principle cell line and the PC3 cells as a relevant prostate cancer model cell line; hence, the HeLa cells were not included in studies from this point onward. PCR integration checks were performed on the 293T and PC3 clonal populations to determine the efficiency of HITI- versus HDR-mediated reporter gene integration at the AAVS1 site (Fig. 2, A and B). The number of 293T clonal populations with correct integration was 11.8% (4 of 34) for HDRgA-engineered cells and 36.1% (13 of 36) for HITIgA clones (Fig. 2, A and B). PC3 cells grew fewer colonies but showed zero integration at the AAVS1 site for tdT⁺ HDR-engineered cells (0 of 14), whereas 10.5% (2 of 19) of the HITI-engineered colonies had correct reporter gene integration, indicating that HITI was more efficient in both cell types.

In vitro reporter gene imaging

Next, we expanded single 293T and PC3 HITIgA clonal cells (with correct integration bands) for further in vitro reporter gene functionality testing. First, we confirmed tdT fluorescence for both the 293T-HITI (Fig. 3A) and PC3-HITI (Fig. 3G) clones via fluorescence microscopy. Next, we confirmed a positive correlation between BLI signal and increasing cell numbers for 293T (Fig. 3, B and C; r² = 0.9718) and PC3 (Fig. 3, H and I; r² = 0.9897) cells. BLI signal measured to at least 10 passages showed stable FLuc2 expression over time for both clonal cell lines (Fig. 3, D and J). To test for Oatp1a1 functionality, 293T naïve, 293-HITI, PC3 naïve, and PC3-HITI cells were incubated with or without Gd-EOB-DTPA (6.4 mM) in normal medium for 90 min, washed thoroughly, pelleted, and inserted into an agarose phantom. Inversion recovery MRI was performed at 3 T, and spin-lattice relaxation rate (R₁) maps were generated for 293T (Fig. 3E) and PC3 (Fig. 3K) cell populations. Neither the naïve 293T/PC3 nor untreated 293T-HITI and PC3-HITI cell populations exhibited any change in R₁ rates (Fig. 3, F and L). Only HITI clones expressing Oatp1a1 had significantly increased R₁ rates after Gd-EOB-DTPA incubation, with ~10-fold increase for 293T-HITI cells (7.952 ± 0.87 s⁻¹) compared with naïve, treated controls (0.806 ± 0.038 s⁻¹; n = 3, P < 0.001; Fig. 3F) and ~5-fold increase for PC3-HITI cells (3.426 ± 0.217 s⁻¹) compared with naïve, treated controls (0.6402 ± 0.045 s⁻¹; n = 3, P < 0.001; Fig. 3L).

Oatp1a1 sensitivity

The MR detection limit of Oatp1a1-expressing 293T and PC3 HITI cell clones was investigated by varying the ratio of naïve:HITI cells in MR phantoms and in vivo with subcutaneous PC3-HITI cell injections. In all instances, MRI and BLI were performed on the same day of the experimental setup. For MR phantoms, all cells (except a naïve-no Gd control, “naïve-NG”) were incubated with Gd-EOB-DTPA (5.2 mM, 60 min), thoroughly washed, and then combined so that each 20 × 10⁶ cell sample would contain a defined number of Oatp1a1-expressing HITI cells and naïve cells (expressed as percent-HITI of the total cell population; see Fig. 4 and fig. S4). The cell pellets were then placed into an agarose phantom, and inversion recovery MRI was performed at 3 T. Spin-lattice relaxation rate (R₁) maps were generated for both PC3 (Fig. 4A) and 293T (fig. S4A) cells. Note that the phantoms in Fig. 4A were rearranged in a linear format for figure presentation only and at two different scales—one on a full scale of R₁ values and the second on a saturated scale (maximum 2.0 Hz as the highest concentration) to show visual differences at the lower concentrations. The change in sensitivity with increasing HITI cell numbers can be clearly seen in stacked histograms showing pixel-by-pixel R₁ counts for each PC3 (Fig. 4B) and 293T (fig. S4B) cell pellet. For both cell types, a significant increase in the average R₁ values, compared to naïve controls, was evident when only 10% of the cell pellet contained Oatp1a1-expressing HITI cells (Fig. 4C for PC3 and fig. S4C for 293T).

We next injected various combinations of PC3-naïve and PC3-HITI cells at five sites subcutaneously on the backs of nude mice to analyze Oatp1-expressing cell detection sensitivity in vivo (Fig. 4, D and E, and fig. S5). A total of 3 × 10⁶ cells were injected per site with the following number of PC3-HITI cells: 0 (naïve cell only control), 10⁴, 10⁵, 10⁶, 3 × 10⁶ (PC3-HITI cell only control). Naïve cells were included with HITI cells so that each injection contained a total of 3 × 10⁶ per site. BLI signal intensity increased as PC3-HITI cell numbers increased (representative mouse shown in Fig. 4D), with 10⁶ and 3 × 10⁶ HITI injections showing significant signal increase above naïve background controls (Fig. 4E). Transverse MR images from the same mouse showed positive contrast at both the 10⁶ and 3 × 10⁶ HITI injection sites 5 hours after Gd-EOB-DTPA injection (Fig. 4F). Similar to the BLI data, these sites also exhibited significantly higher contrast-to-noise ratios (CNRs) than naïve controls (Fig. 4F). The 10⁴ and 10⁵ PC3-HITI injections were difficult to visualize on MRI as the phosphate-buffered saline (PBS) of these injections spread out over the ~6 hours after injection and had no discernible positive contrast, so they could not be measured. These data were consistent across all three mice (see fig. S5) and showed that the very minimum number of Oatp1a1-expressing cells we could detect with Gd-EOB-DTPA–based MR contrast was 10⁶ cells in a 50-μl subcutaneous injection volume.

Effect of Gd-EOB-DTPA on HITI-engineered Oatp1a1-expressing cells

Trypan blue exclusion assays and cell counts were performed to determine whether the uptake of Gd-EOB-DTPA into Oatp1a1-expressing 293T and PC3 cells affected cell viability and growth over time. Cells were plated (day 0) and incubated with 5.2 mM Gd-EOB-DTPA contrast agent or saline the next (day 1) for 90 min. There was no significant difference in viability or growth rate between 293T-naïve and 293T-HITI cells or between control (saline) or Gd-EOB-DTPA–treated cell groups (fig. S6A). In contrast, the PC3-HITI clone exhibited a slower growth rate compared to the mixed population of naïve cells (fig. S6B). Exposure to Gd-EOB-DTPA, however, did not affect the viability or growth of either cell type compared to their no-Gd (saline) controls (fig. S6B). These data indicate that Gd-EOB-DTPA uptake had no negative effect on the cells. However, the single PC3-HITI clone we obtained was slower growing compared to the mixed cell naïve population.

PC3-HITI Oatp1a1 tumor models for MRI detection

As a proof of principle that our HITI-engineered cells could show Gd-EOB-DTPA–induced positive MRI contrast in subcutaneous tumors, we injected 293T-naïve and 293T-HITI cells on either flank of a nude mouse (fig. S7). For both cell types, the large masses were visible on precontrast images and showed noticeable positive contrast 20 min after Gd-EOB-DTPA injection. However, 5 hours after contrast, the naïve tumor had returned to precontrast background levels, whereas the HITI tumor had very prominent positive contrast that also showed heterogeneity within the tumor mass (fig. S7). This heterogeneity, in contrast, likely reflects areas of viable (enhancing) and nonviable (non-enhancing) Oatp1-expressing cells within the tumors, as we have reported previously (45).

Moving into a more relevant cancer model, we next injected PC3-naïve and PC3-HITI clonal cells subcutaneously on either flank of nude mice and followed BLI and MRI signal changes over time (Fig. 5). At only 11 days after injection, before the tumors were visible or palpable, clear positive contrast was observed for HITI-engineered cells 5 hours after Gd-EOB-DTPA injection, whereas naïve tumors were undistinguishable (Fig. 5A and fig. S8). The same mice were then imaged at day 46, where the naïve tumor was visible due to pooled Gd-EOB-DTPA at 20 min after contrast injection. In a similar fashion to 5 hours after contrast, only the HITI-engineered cells retained the Gd agent and showed bright, positive contrast (Fig. 5B and fig. S9). Contrast-to-noise measurements, tumor size, and estimated cell numbers for PC3-HITI tumors all increased from days 11 to 46 (Fig. 5, C to E). These data suggest that the Oatp1a1 MRI reporter can detect tumor burden at stages where the tumors are not visible or palpable, and tumor growth can be tracked longitudinally with Gd-EOB-DTPA–enhanced MRI.

Last, PC3-HITI cells were injected via the tail vein into immunocompromised NOD scid gamma (NSG) and nude mice to investigate the ability of Oatp1a1 as a reporter gene for visualizing metastases (Fig. 6 and fig. S10). Using BLI as a guide, we were able to detect metastatic tumors in the head of NSG (Fig. 6) and nude (fig. S10) mice, 84 and 114 days after injection, respectively. Tumors were undistinguishable in precontrast MR images for the NSG mouse. However, 5 hours after Gd-EOB-DTPA injection, a cluster of small, enhanced PC3-HITI tumors were clearly visible (yellow arrows, Fig. 6), which equated to an overall tumor size of 2.05 mm³ and, assuming that a 1-cm³ tumor contains around 1 × 10⁹ cells, contained around 2.1 × 10⁶ cells. Similarly, in the nude mouse model, a larger, more defined tumor showed strong enhancement 5 hours after contrast injection (fig. S10), which measured 31.42 mm³ in size and contained ~3.1 × 10⁷ cells. These data indicate the usefulness of this reporter gene for detecting metastatic burden with contrast-enhanced MRI.

DISCUSSION

As personalized medicine and CRISPR-editing become a reality in the clinic, there is a greater need to (i) improve the efficiency, efficacy, and safety of genetically engineered cell therapies, and (ii) improve our understanding of disease progression and treatment response in preclinical models of disease. Reporter gene-based imaging allows us to track the location, viability, growth, and efficacy of such treatments, and in preclinical models of cancer progression and treatment. In this study, we have developed a non–viral vector–based engineering system for large DNA multimodality reporter gene integration into the AAVS1 safe harbor genomic locus. To improve safety further, we used MCs as the DNA vector of choice, which eliminates bacterial DNA contamination and antibiotic resistance genes. In addition, we showed that using the NHEJ repair pathway with HITI could improve DNA editing efficiency in human cells compared to the more commonly used HDR pathway. Last, building off our previous work (34), we have engineered a trimodality reporter gene construct that contains a clinically relevant MRI reporter, Oatp1a1, in addition to fluorescent and bioluminescent genes, which enabled cell sorting and noninvasive BLI/MRI of engineered cells in a preclinical cancer model.

One of the major limitations of engineering cells with large, multimodality reporter gene DNA plasmids is the reduced efficiency of both transfection and gene editing with increasing construct/insert size (46–48). In addition, the presence of bacterial and antibiotic resistance genes in PPs has the potential to exert immunological responses and raises safety concerns. To circumvent these issues, we designed our study to use MCs, which remove the bacterial backbone from PPs and thus reduce the size of the DNA donor constructs. Using MCs instead of PPs allowed us to remove ~4 kb of unwanted DNA from our HDR construct, with a further reduction of ~1.5 kb for the HITI MC when the homologous arm sequences were replaced with a 20-bp gRNA sequence (saving a total of ~5.5 kb). These large-scale reductions thus provided us with room to upgrade our dual-modality tdTomato and FLuc2 reporter gene construct we previously reported (34) to a trimodality reporter gene construct with the addition of the Oatp1a1 MRI reporter (44, 45). To improve safety and translatability, we also removed the puromycin resistance gene to reduce the MC size by a further 600 bp and used FACS of tdTomato-positive cells to obtain mixed and clonal cell populations instead of antibiotic selection. Our final step for improving safety was to design our system to target a “safe harbor” locus in genomic DNA. Several of these loci have now been reported in the literature (49) and are described as sites where inserted genetic elements can function as intended, without causing alterations that would pose a risk to the host cell or organism (25). For this study, we targeted the AAVS1 site found within the human protein phosphatase 1 regulatory subunit 12C (PP1R12C) gene, as this has been one of the best characterized, to date. No known side effects are associated with disrupting the PP1R12C gene; however, it has been reported that mechanisms such as DNA methylation can silence transgenes targeted to this genomic region (50). Because our studies rely on stable reporter gene expression over time for accurate cell detection and proliferation, we investigated whether reporter gene expression in our AAVS1-engineered 293T-HITI and PC3-HITI cell populations changed over time. We found that BLI signal was stable to at least 10 passages and tdT fluorescence was expressed in both cell lines, indicating consistent transgene expression.

We have shown here that HITI-based CRISPR-Cas9 cell engineering is more efficient than the more commonly used HDR method for integrating large DNA donor constructs. Targeted transgene integration is typically achieved using homologous arms and the HDR pathway; however, this mechanism is highly inefficient and not usually active in nondividing cells (35). Our previous study showed that only 3.8% of selected cells were correctly edited using the HDR mechanism (34). In contrast, the HITI method that uses the NHEJ pathway is active in all stages of the cell cycle and in quiescent cells (38) and thus has been used to improve editing efficiency. Using the method described by Suzuki et al. (40), our engineered 293T and PC3 clonal cell populations did have greater DNA integration at the AAVS1 site compared with HDR (36 and 12% for HITI versus 10.5 and 0% for HDR, respectively). However, the NHEJ repair pathway is error prone and often leads to insertions and deletions (indels) at the DNA junctions. Consequently, this mechanism is often taken advantage of to produce DNA disruptions, gene silencing, and knockouts. These issues would need to be considered if using the HITI method for correctional DNA editing and promoter-less vector integration, because these require specific DNA sequences, either upstream or downstream, to be preserved. In this case, we engineered cells with the only requirements being that the transgene inserts into the AAVS1 site (confirmed with junctional PCR) and that the reporter genes are consistently expressed (confirmed with imaging). Therefore, indels at either the 5′ or 3′ junction would likely have a negligible impact on our experiments.

Although we confirmed correct transgene integration at the AAVS1 site in our study, we cannot rule out integration at other off-target sites in HITI- and HDR-engineered populations. Several 293T and PC3 single-cell clonal populations expressed the tdTomato fluorescence reporter gene but did not show integration bands for the AAVS1 site. Evidence suggests that CRISPR-Cas9 is not 100% accurate and off-target effects have been reported as a common problem associated with CRISPR (51, 52). Thus, it is likely that some MCs integrated into off-target Cas9 cut sites in clones where the correct AAVS1 integration bands were absent or that the MCs inserted into the AAVS1 site in the wrong direction. Although HITI is designed to minimize integration in the wrong orientation, the error-prone NHEJ repair mechanism of blunt-ended DNA breaks could lead to indels at the CRISPR-Cas9 cut site boundaries, which could then disrupt the ability of Cas9 to recognize and recut those sites. Although our preliminary data suggest that stable integration in the wrong direction with HITI was absent in our sorted MCPs, there remains a possibility of indel formation using Cas9 HITI. This could be reduced in future studies by adopting a similar method to that recently reported by Li and colleagues (53), where 5-bp overhangs created by Cas12a could lead to more precisely edited genomes, in a process coined microhomology-dependent TI (MITI) (53). Independently of CRISPR, MCs, like plasmids, can also randomly integrate into the genome of cells, albeit at very low rates. Future work will need to analyze the rate of off-target integrations and possible indel disruptions at the CRISPR-Cas9 cut sites using techniques, such as next-generation sequencing, to determine the full safety profile of HITI at safe harbor loci. To improve targeting specificity, studies have shown that high-fidelity Cas9 enzymes in ribonucleoprotein complexes (RNPs), instead of Cas9 DNA vectors, improve on-target activity while reducing off-target editing (54, 55). In combination with RNPs, AAVs are now commonly used as DNA donors for CRISPR experiments due to their high transduction capabilities in hard-to-transfect cell lines, their low risk of random integration, and reduced immunogenic response. However, AAVs are still limited by their loading capacity of ~4.5 kb, which would be a problem for large, multimodality imaging vectors as presented here, but conceivable for future studies where only one imaging reporter gene is required. With these emerging technologies, it is likely that CRISPR gene editing will become highly specific and thus safer in the near future.

We engineered cells with a multimodality reporter gene construct to enable us to go from single cell, optical imaging methods (FLI) to higher-sensitivity whole-animal planar imaging (BLI) and superior 3D high-resolution tomographic imaging (MRI) in animals. This offers several advantages. First, fluorescently activated cell sorting of tdTomato-expressing cells eliminates the need for an antibiotic resistance selection gene, which constitutes a safety risk and has been associated with structural plasmid instabilities (56). Second, the firefly luciferase gene (FLuc2), in combination with its substrate D-luciferin, allows us to directly visualize engineered cells in vivo using BLI. Inclusion of bioluminescent genes in preclinical cancer models is a relatively inexpensive and valuable tool that also allows one to track cell migration and cell seeding in metastatic cancer models, assess cell viability, and follow cell/tumor growth longitudinally (14). A limitation of BLI is that it is restricted to small animal models of disease. However, it is useful for determining sites of cell arrest/seeding/growth and thus can be used in conjunction with other reporter genes as a guide for determining when and where to perform relatively more expensive, higher-resolution clinical imaging, such as MRI (13). To build off our previous dual FLI-BLI study (34), we decided to include the MRI reporter gene, Oatp1a1, as a translationally relevant and sensitive reporter gene to complete our trimodality construct for HITI-based CRISPR engineering. First described by Patrick et al. (44), Oatp1a1 selectively, but reversibly, uptakes the clinically approved Gd³⁺ contrast agent Gd-EOB-DTPA and thus provides positive contrast in T₁-weighted MR images. The authors concluded, therefore, that Oatp1a1-engineered cells and tumors should be easier to detect than the negative contrast generated by T₂ agents, such as superparamagnetic iron oxide (SPIO) and ferromagnetic agents (44, 57). In addition, engineering cells with integrated Oatp1a1 expression means that MR images can be obtained longitudinally to track cell migration and growth, and signal intensity can be directly correlated with cell viability. Last, we and others have found that Oatp1a1 also enhances the uptake of D-luciferin for BLI (45, 58) and the fluorescent dye indocyanine green (using the human ortholog OATP1B3) for both fluorescent (59) and photoacoustic imaging (60), which gives an added advantage of using Oatp1 for multimodality imaging. Because we, and others, have now shown that the human OATP1B3gene also functions as a useful fluorescent, photoacoustic, and MRI reporter gene, in vivo (59, 60), future studies will focus on exchanging Oatp1a1 for the more translationally favorable OATP1B3 ortholog.

In this study, we set out to determine the smallest number of Oatp1a1-expressing HITI-engineered cells that we could detect using MRI phantoms and in vivo injections (with BLI as a guide). Our in vitro MRI phantom experiments showed significant increases in R₁ rates when 10% of the cell pellet, and thus only 10% of each voxel, contained Oatp1a1-expressing cells. For in vivo sensitivity measurements, we were able to reliably detect 1 million cells in a 50-μl volume. The cell and contrast injections and imaging were all performed on the same day so that the cell numbers would not vary with migration, cell death, or proliferation. Similar experiments have been performed for PET reporter genes. However, there are some issues with this experimental setup as we prepared cells in PBS rather than within a matrix such as Matrigel so as not to impede diffusion of the contrast agent. These were merely subcutaneous injections of a known number of cells in a known volume of PBS, not tumors. Without time to develop blood vessels, as you would with tumors, it is likely that some of the contrast agent could not diffuse into the injection site fully. This may explain why the contrast enhancement appears as a ring around the cell injection sites. We also believe that this problem likely contributed to the lack of signal enhancement in the 10⁴ and 10⁵ injection sites, which without a matrix to hold them in place also had the problem of cell spreading over a larger surface area. In summary, there are no easy ways to measure the sensitivity of exact cell numbers in vivo, especially with a reporter gene system that relies on an injected substrate; however, the combination of in vitro and in vivo experiments give us a better idea of the minimum number of cell numbers we can confidently detect.

When expressing reporter genes in cells, it is important to know whether the protein and/or its substrates affect the normal function or viability of the cell. In our study, we noticed that the Oatp1a1-expressing PC3-HITI cells grew slower compared to the mixed cell naïve population in vitro and in vivo, whereas their 293T-HITI counterpart showed no difference compared to naïve cells. Although unfortunate for comparative purposes, we believe that the slow growth rate of the PC3-HITI cells was simply due to heterogeneity in clonal populations—especially when comparing to mixed naïve populations—and not due to expression of Oatp1a1. Our data also support this because the 293T-HITI cells were not affected. In addition, we and others have already shown that there is no impact of Oatp1a1 on cell growth in several different cell types (44, 45). Ideally, several correctly engineered clones would be combined for biological and imaging experiments. We were limited in this case by obtaining just one usable PC3-HITI clone. However, the clone still grew primary and metastatic tumors, which we could sensitively detect with BLI and MRI. In addition, the uptake of Gd-EOB-DTPA did not have any detrimental impact on cell viability or growth, indicating that Oatp1 is a viable option for reporter gene imaging and cell tracking.

The improved safety profile and expression of multimodal reporter genes proposed here could have several uses in cell engineering, or at least help answer several concerns with in vivo cell therapies. For example, the U.S. Food and Drug Administration have listed potential safety concerns related to unproven stem cell therapies (61), including (i) the ability of cells to move from placement sites and change into inappropriate cell types or multiply, (ii) failure of cells to work as expected, and (iii) the growth of tumors. In addition, the long-term safety profiles of cells engineered with randomly integrating viruses still require further investigation and optimization. These are concerns that could be addressed by targeting nonviral DNA vectors, such as MCs, to specific safe harbor loci, such as AAVS1, and reducing the use of integrating viruses. Incorporating reporter genes for clinical grade imaging will also help improve patient safety by allowing one to track cellular therapies in vivo [such as for stem cells or cancer-homing theranostic cells (62)]. Clinicians could then determine whether the therapeutic cells are localizing to the correct anatomical feature, such as a solid tumor (18), or to determine their persistence and viability for short- and long-term treatment strategies. Future work will focus on evaluating our system in stem cells and other clinically relevant cell types. Translation will also need to consider building donor vectors that lack optical reporter genes and use other selection methods (e.g., magnetic sorting). It is easily feasible to switch out genes from our trimodality construct for other imaging purposes, such as replacing FLuc2 with a PET reporter gene for dual PET-MR imaging. Suicide switch genes could also be incorporated to further improve safety by killing the engineered cells in cases where they become oncogenic (63), for example. These tools not only are useful for clinical cell-based therapies but also are extremely useful in preclinical studies for investigating cancer progression/aggression, metastatic burden, and treatment strategies. Avoiding the use of random-integrating viruses and targeted editing should also help reduce off-target effects of gene editing that may alter the normal characteristics of the cell type being studied.

CONCLUSION

Our work demonstrates the first CRISPR-Cas9 HITI MC system for safe harbor integration of a large donor construct encoding three reporter genes for multimodal longitudinal imaging of cells in vivo. We have shown that inclusion of the translationally relevant MR reporter gene, Oatp1a1, can enable localization and tracking of small primary and metastatic tumors that are not readily detectable visually or in precontrast MR images. This work lays the foundation for an effective and safer nonviral genome editing tool for noninvasive reporter gene tracking of multiple cell types in vivo.

MATERIALS AND METHODS

Constructs

Construct designs are shown in Fig. 1B. The pCas9-AAVS1guideRNA-zsG-MC (Cas9-AAVS1-MC) and pCas9-scrambledRNA-zsG-MC (Cas9-scrambled-MC) PPs originated from pCas-Guide-AAVS1 and pCas-Guide-Scrambled plasmids purchased from Origene (MD, USA). The Cas9 enzyme and gRNA sequences were cloned between attB and attP recombination sites in an MC bacterial backbone containing a ZsGreen (zsG) fluorescence reporter driven by the elongation factor 1-α promoter (hEF1α). The AAVS1-HDR-tdT-Fluc2-Oatp1a1-MC (HDR-MC) construct was derived from an HDR vector lacking the Oatp1a1 gene as we described previously (34). This plasmid is driven by the hEF1α promoter and expresses tdTomato (tdT), firefly luciferase (Fluc2), and organic anion transporting polypeptide 1a1 (Oatp1a1) using a self-cleaving 2a peptide system. For improved expression, the plasmids also contain the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) followed by the human growth hormone polyadenylation signal (hGH polyA). The HDR plasmid contains the left and right homologous arms (RHA: 527 bp, LHA: 481 bp) that are complementary to the region flanking the AAVS1 cut site; the homologous arms were obtained from the pAAVS1-puroDNR plasmid from Origene (MA, USA). The Oatp1a1 gene was added through PCR amplification from a previously made vector we constructed using PGK_Straw_E2A_Oatp1a1 (a gift from K. Brindle’s laboratory; University of Cambridge). Using the HDR-MC PP as the template, we generated the pAAVS1-HITI-tdT-Fluc2-Oatp1a1-MC (HITI-MC) PP using the In-Fusion Cloning Kit from Clontech (Takara Bio, CA, USA). Using restriction enzyme digestion, we extracted the bacterial backbone and MC recombination sites and then extracted the three reporter genes (without the homologous arms)—tdT, Fluc2, and Oatp1a1—from the HDR-MC construct using PCR. However, for HITI functionality, we designed our primers to also include a 23-bp extension (5′-GTTAATGTGGCTCTGGTTCTGGG-3′) downstream of the polyA sequence, which incorporates the same cut site and protospacer adjacent motif (PAM) sequence for our AAVS1 gRNA, which allows Cas9 cutting of both the MC and genomic DNA.

MC production

ZYCY10P3S2T Escherichia coli (System Biosciences, Palo Alto, CA, USA) were transformed with the original PPs of all four constructs (HDR-MC or HITI-MC, Cas9-AAVS1-MC, and Cas9-scrambled-MC), and viable colonies were selected using kanamycin plates. Colonies were picked 24 hours after transformation and grown in 6 ml of lysogeny broth (LB) with kanamycin for 6 hours at 37°C, followed by growth in terrific broth (TB) for 12 hours at 37°C. To induce expression of the phiC31 integrase for MC production via attB and attPrecombination, 100 ml of LB broth together with 100 μl of 20% arabinose induction solution (System Biosciences, Palo Alto, CA, USA) and 4 ml of 1 N NaOH was added to the culture and grown for 5.5 hours at 30°C. An endotoxin-free maxi kit (Qiagen, Valencia, CA, USA) was used to purify both PP and MC. Following purification of the MC products, PP contamination was removed using the Plasmid Safe ATP-dependent DNase Kit (Epicentre, WI, USA), and the products were cleaned and concentrated using the Clean & Concentrator-25 Kit (Zymo Research, CA, USA).

Cell culture and transfection

HEK-293T cells and human adenocarcinoma HeLa cells (both from the American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (Wisent Bioproducts, Québec, Canada) supplemented with 10% fetal bovine serum (FBS; Wisent Bioproducts, Québec, Canada) and 1× antibiotic-antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). Human grade 4 adenocarcinoma PC3 cells were a gift from H. Leong (Western University, ON, Canada) and were grown in RPMI (Wisent Bioproducts, Québec, Canada) supplemented with 5% FBS and 1× antibiotic-antimycotic. Cells were transfected with the linear polyethylenimine transfection agent jetPEI (Polyplus-transfection, Illkirch, France), according to the manufacturer’s instructions. Briefly, cells were grown in six-well plates until 80 to 90% confluency and cotransfected with 1 μg each of Cas9-AAVS1-MC or Cas9-Scrambled-MC together with 1 μg of the donor MC constructs: HDR-MC or HITI-MC, for a total DNA mass of 2 μg. The DNA was prepared in 150 mM NaCl and complexed with 4 μl of jetPEI reagent per well.

FACS and flow analysis

All FACS and flow cytometry was performed at the London Regional Flow Cytometry Facility (Robarts Research Institute, London, Canada). Forty-eight hours after transfection, the population of cells displaying both red (tdTomato) and green (zsGreen) fluorescence were sorted using a BD FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA). At selected time points following FACS, the cells were analyzed for tdTomato fluorescence using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA). Either 14 or 21 days after the initial sort, the cells were again sorted on the FACSAria III to purify tdTomato-positive cells only (referred to as the pooled population). In this regard, our protocol aimed to sort cells that had incorporated the MC inserts (based on tdTomato fluorescence) into the genome and excluded any cells that had randomly integrated Cas9 MC DNA (zsGreen). At the same time as the second (tdTomato) sort, individual cells were plated into wells of a 96-well plate to enable single-cell colonies to be grown and expanded (referred to as clonal cell populations).

Genomic DNA extractions and AAVS1 integration analysis

Extraction of genomic DNA from the pooled population of cells was performed using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. DNA quality and concentrations were measured on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Extraction of genomic DNA from clonal populations was performed as we described previously (34). Briefly, cell pellets were resuspended in a QuickExtract DNA extraction solution (Lucigen, Middleton, WI, USA), incubated at 65°C for 10 min, vortexed, and incubated at 98°C for 5 min. The DNA was then directly used for PCR or stored at −20°C. To check for integration at the AAVS1 site, two primers were designed to amplify the 3′ junction between the donor cassette and the AAVS1 site outside of the homologous arm region. The forward primer was uniquely complementary to the polyA tail in the MC cassette (5′-CCTGGAAGTTGCCACTCCAG-3′) and the reverse primer to the AAVS1 site (5′-AAGGCAGCCTGGTAGACAGG-3′). A 1.3-kb PCR product was produced if the MC-HDR was correctly integrated at the AAVS1 site, and a 1.7-kb PCR product was produced if MC-HITI was correctly integrated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were designed as DNA loading controls and to confirm successful DNA extractions: forward 5′-TTGCCCTCAACGACCACTTT-3′ and reverse 5′-GTCCCTCCCCAGCAAGAATG-3′ and yielded a PCR product of 502 bp. Agarose gel electrophoresis with 1% agarose gels and RedSafe (FroggaBio, ON, Canada) was used to separate and visualize PCR products.

In vitro fluorescence and BLI

The pooled and clonal cell populations were evaluated for tdTomato fluorescence expression on an EVOS FL auto 2 microscope (Thermo Fisher Scientific, Waltham, MA, USA). For BLI experiments, varying cell numbers were plated in triplicate into black walled 96-well plates. D-Luciferin (0.1 mg/ml; PerkinElmer, Waltham, MA, USA) was added to each well, and images were rapidly collected on the IVIS Lumina XRMS In Vivo Imaging System (PerkinElmer) equipped with a cooled charge-coupled device (CCD) camera. Average radiance values in photons s⁻¹ cm⁻² sr⁻¹ were measured from regions of interest drawn around each well using LivingImage software (PerkinElmer).

In vitro MRI

Naïve and Oatp1a1-expressing cell clones were seeded in 15-cm tissue culture dishes and grown to confluency. Cells were incubated with medium containing 5.2 mM Gd-EOB-DTPA or with medium containing an equivalent volume of PBS for 90 min at 37°C and 5% CO₂. Cells were then washed three times with PBS, trypsinized, and pelleted (20 × 10⁶ cells per pellet) in 0.2-ml Eppendorf tubes. For sensitivity experiments, various numbers of naïve:HITI cells were combined in Eppendorf tubes, mixed well, and then pelleted. The tubes were placed into a 1% agarose phantom mold, and MRI was performed on a 3-T GE clinical MRI scanner with an eight-channel head RF coil (General Electric Healthcare Discovery MR750 3.0 T, Milwaukee, WI, USA). A fast spin echo inversion recovery (FSE-IR) pulse sequence was used with the following parameters: matrix size, 256 × 256; repetition time (TR), 5000 ms; echo time (TE), 16.3 ms; echo train length (ETL), 4; number of excitations (NEX), 1; receiver bandwidth (rBW), 25 kHz; inversion times (TIs), 25, 50, 100, 200, 350, 500, 750, 1000, 1500, 2000, 2500, and 3000 ms; in-plane resolution, 0.27 mm × 0.27 mm; slice thickness, 2.0 mm; scan time, 5 min and 25 s per TI. Spin-lattice relaxation rates (R₁) were determined by nonlinear least-squares fitting (MATLAB, MathWorks, Natick, MA, USA) of the following equation to the signal intensity across the series of TIs on a pixel by pixel basisS=∣Mss−(Mss−Mi)·e−TIT1/∣

Here, S, M_ss, and M_i represent the acquired signal, the longitudinal magnetization in steady-state equilibrium, and the initial longitudinal magnetization acquired after the inversion pulse, respectively. T₁ is the spin-lattice relaxation time such that R1=T−11 and TI is the inversion time.

Animal models

All animal protocols were approved by the University Council on Animal Care at the University of Western Ontario (protocol #2015-058) and follow the Canadian Council on Animal Care (CCAC) and Ontario Ministry of Agricultural, Food and Rural Affairs (OMAFRA) guidelines. Crl:NU-Foxn1^nu (nude) male mice (Charles River Laboratories, Wilmington, MA, USA; N = 3 to 5) aged 6 to 8 weeks were used for subcutaneous and metastatic tumor model injections, and NOD.Cg-Prkdc^scid Il2rg^tm1WjI/SzJ (NSG) immunodeficient male mice (obtained from the Humanized Mouse and Xenotransplantation Facility at the Robarts Research Institute, University of Western Ontario, London, Canada; N = 3) were used for experimental metastasis models (intravenous cell injections).

In vivo BLI

BLI was performed on the same IVIS Lumina XRMS system described for in vitro imaging. Mice were anesthetized with 2% isoflurane in 100% oxygen using a nose cone attached to an activated carbon charcoal filter for passive scavenging and kept warm on a heated stage. Anesthetized mice received a 100-μl intraperitoneal injection of D-luciferin (30 mg/ml), and BLI images were acquired with automatic exposure times until the peak BLI signal was obtained (up to 40 min). Regions of interest were manually drawn using LivingImage software to measure average radiance (photons s⁻¹ cm⁻² sr⁻¹). The peak average radiance was used for quantification for each mouse.

In vivo MRI and quantification

All mouse MRI scans were performed with a custom-built gradient insert and a 3.5-cm-diameter birdcage RF coil (Morris Instruments, Ottawa, ON, Canada), as we described previously (45). Mice were kept anesthetized during the scan with 2% isoflurane administered via a nose cone attached to the coil. T₁-weighted images were acquired using a 3D spoiled gradient recalled acquisition in steady-state pulse sequence using the following parameters: field of view, 50 mm; TR, 14.7 ms; TE, 3.3 ms; rBW, 31.25 MHz; matrix size, 250 × 250; flip angle, 60°; NEX, 3; 200-μm isotropic voxels; scan time, approximately 15 min per mouse. Precontrast images were acquired followed by administration of Gd-EOB-DTPA (1.67 mmol/kg) (Primovist; Bayer, Mississauga, ON, Canada) via the tail vein. Mice were then reimaged 20 min later for immediate postcontrast images, which provide positive contrast to many tissues, including the naïve tumors, as a result of Gd-EOB-DTPA pooling, and/or 5 hours later for Oatp1a1-specific uptake. This time point was determined to allow enough time for Gd-EOB-DTPA to be cleared, yet still provided strong positive contrast in Oatp1a1-expressing cells (44, 45). CNR and tumor size measurements were calculated from MR images using ITK-snap open source software (www.itksnap.org) (64). Tumors were manually segmented in three dimensions by tracing the tumor or control tissue (hind leg muscle) with polygon and paintbrush tools and pixel intensity recorded in every slice. The CNR of tumors was calculated by taking the signal intensity of the difference between tumor regions and muscle tissue divided by the SD of background signal(CNR=attenuationtumor−attenuationmuscle Std.Dev.background)

The number of cells in the tumors was estimated from the assumption that a tumor reaching a size of 1 cm³is estimated to contain around 1 × 10⁹ cells.

In vivo Oatp1a1-induced Gd-EOB-DTPA uptake MRI and BLI sensitivity

To evaluate the cellular detection sensitivity of Oatp1a1-expressing cells with Gd-EOB-DTPA–enhanced MRI, nude mice were injected with 50 μl of cell suspensions in PBS containing 3 × 10⁶ total cells per injection at the following ratios: 3 × 10⁶ naïve cells alone; 10⁴ PC3-HITI + 2.99 × 10⁶ naïve cells; 10⁵ PC3-HITI + 2.9 × 10⁶naïve cells; 10⁶ PC3-HITI + 2 × 10⁶ naïve cells; and 3 × 10⁶ PC3-HITI cells alone, subcutaneously in five locations on the back/flank region. Immediately after cell injections, Gd-EOB-DTPA (1.67 mmol/kg) was injected into the tail vein, and mice were imaged on a 3-T clinical grade MR scanner 5 hours later. This time point allows clearance of Gd-EOB-DTPA from the body yet provides sufficient time for the agent to penetrate the subcutaneous injections sites and accumulate in Oatp1a1-expressing cells. After MRI, mice were moved to the IVIS scanner and injected with 100 μl of D-luciferin (30 mg/ml) intraperitoneally and BLI was performed, as described earlier.

293T and PC3 tumor models

293T or PC3 naïve and HITI-engineered cells were injected subcutaneously (2.5 × 10⁶ 293Ts and 1 × 10⁶PC3s) on the left and right flanks of nude mice, respectively (293T, N = 2; PC3, N = 5). For experimental metastasis studies, 5 × 10⁵ PC3 naïve or HITI-engineered cells were injected into the tail veins of NSG or nude mice (N = 3). Tumor growth was tracked on a weekly basis with BLI, as described above. MRI was performed on mice at various time points, as indicated in the results section. First, a precontrast scan was performed on all mice, followed immediately with injection of the Gd-EOB-DTPA contrast agent into the tail vein (1.67 mmol/kg). For some experiments, the mice were rescanned 15 to 20 min after contrast injection to show tumor and whole-body distribution of Gd-EOB-DTPA. In all instances, MRI scans were performed ~5 hours after contrast injection because Oatp1a1-expressing cells still retain Gd-EOB-DTPA and show strong positive contrast at this time point. This also allows enough time for washout of Gd-EOB-DTPA in most tissues and organs (except for the gastrointestinal tract and bladder where cleared Gd-EOB-DTPA accumulates before being excreted) (44).

Statistical analysis

Statistical analysis was performed with GraphPad Prism version 7 (GraphPad Software Inc., CA, USA; www.graphpad.com) software. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used for in vitro and in vivo BLI and CNR data analysis. An unpaired one-tailed t test with Welch’s correction was used to analyze the increase in CNR/cell numbers for PC3-HITI day 11 versus day 46 tumors.

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/7/4/eabc3791/DC1

View/request a protocol for this paper from Bio-protocol.https://creativecommons.org/licenses/by-nc/4.0/

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

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Acknowledgments: We thank K. Brindle (University of Cambridge) for providing the Oatp1a1 plasmid, Y. Xia for help with paper revisions, K. Chadwick for FACS and flow cytometry help, and D. Reese for MRI troubleshooting. Funding: This work was funded by a Canadian Institutes of Health Research (CIHR) project grant (grant #377071, J.A.R.), a Natural Sciences and Engineering Research Council (NSERC) discovery grant (grant #RGPIN-2016-05420, J.A.R.), and a University of Western Ontario Strategic Support for CIHR Success Seed grant (J.A.R.). Author contributions: J.A.R. designed the project. J.A.R., J.J.K., and M.S.-M. designed the experiments. J.J.K. directed the study and, with M.S.-M., carried out most of the experiments. N.N.N. developed the methods for Oatp1a1 MRI, and F.M.M. developed the MATLAB scripts for processing MRI phantom data. Y.C. helped perform MRI. M.M.E. processed and analyzed MRI data. A.M.H. helped develop the PPs. J.J.K. wrote the manuscript with help from M.S.-M., which J.A.R. reviewed and edited. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

• Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

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Development of optically controlled “living electrodes” with long-projecting axon tracts for a synaptic brain-machine interface

1. View ORCID ProfileDayo O. Adewole¹,²,³,⁴, 
2. Laura A. Struzyna¹,²,³,⁴, 
3. View ORCID ProfileJustin C. Burrell¹,²,³, 
4. James P. Harris¹,², 
5. Ashley D. Nemes¹,², 
6. Dmitriy Petrov¹,², 
7. View ORCID ProfileReuben H. Kraft⁵, 
8. View ORCID ProfileH. Isaac Chen¹,², 
9. Mijail D. Serruya²,⁶,⁷,⁸, 
10. View ORCID ProfileJohn A. Wolf¹,² and 
11. View ORCID ProfileD. Kacy Cullen¹,²,³,⁴,*

 See all authors and affiliationsScience Advances  22 Jan 2021:
Vol. 7, no. 4, eaay5347
DOI: 10.1126/sciadv.aay5347

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Abstract

For implantable neural interfaces, functional/clinical outcomes are challenged by limitations in specificity and stability of inorganic microelectrodes. A biological intermediary between microelectrical devices and the brain may improve specificity and longevity through (i) natural synaptic integration with deep neural circuitry, (ii) accessibility on the brain surface, and (iii) optogenetic manipulation for targeted, light-based readout/control. Accordingly, we have developed implantable “living electrodes,” living cortical neurons, and axonal tracts protected within soft hydrogel cylinders, for optobiological monitoring/modulation of brain activity. Here, we demonstrate fabrication, rapid axonal outgrowth, reproducible cytoarchitecture, and simultaneous optical stimulation and recording of these tissue engineered constructs in vitro. We also present their transplantation, survival, integration, and optical recording in rat cortex as an in vivo proof of concept for this neural interface paradigm. The creation and characterization of these functional, optically controllable living electrodes are critical steps in developing a new class of optobiological tools for neural interfacing.

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INTRODUCTION

Most devices for neuromodulation (e.g., deep brain stimulation electrodes for Parkinson’s disease) and neural recording [commonly called brain-computer interfaces (BCIs)] work by electrically stimulating or capturing neuronal activity within the brain (1). These neural interfaces have been developed across a range of medical applications; two notable milestones include cochlear prostheses for people with hearing loss and thought-driven computer control for people with neuromuscular disorders (1). Despite these achievements, the clinical impact of more advanced neural interfaces is beset by several underlying functional challenges. Implantable BCIs primarily use inorganic microelectrodes, which often exhibit diminished recording quality over time due to a host of biological factors (e.g., inflammation, neuronal loss, and glial scarring) and abiotic biostability issues (including decreasing impedance due to loss of insulation and mechanical failure) (1–5). In parallel, the effectiveness of electrical neuromodulation is limited by an inability to target specific neurons or neuronal subtypes (e.g., excitatory versus inhibitory neurons) within the volume of charge injection, as well as the thresholds for both safe and functional therapeutic stimulation (6). Specificity in neuromodulation may be improved with optogenetics, where inducing the expression of light-sensitive proteins in specific neuronal populations allows these subgroups to be controlled on a wavelength-specific basis through photostimulation. However, the longevity and immune response to viral optogenetic transduction in humans is currently unknown, with nonhuman primate studies suggesting an elevated immunogenic response (7). Further, light scattering properties of tissue block precise photostimulation of neurons more than a few hundred microns deep (8). Deeper tissue is accessible with implantable optical fibers, lenses, or micro–LEDs (light-emitting diodes), yet chronic performance must also address complications from the foreign body response to these materials and overheating of surrounding tissue (9, 10). Across electric and/or optical input-output paradigms, the information transfer bandwidth limits the quality of the neural interface. The ability to address these design challenges—compatibility with the brain, target specificity, and long-term stability—will direct the utility and clinical translation of future neuromodulation and neural recording technologies.

We are advancing a potential solution to these limitations through our existing platform, the microtissue engineered neural network (μTENN). μTENNs are discrete population(s) of neurons connected by long bundles of axons protected within a microscopic hydrogel cylinder (“microcolumn”) (Fig. 1) (11, 12). μTENNs were originally developed to reconstruct lost or injured neuroanatomy by approximating the brain’s network-level structure—locally connected neurons spanned by dense axonal tracts—and previously demonstrated neuronal survival, maintenance of axonal architecture, and synaptic integration with host neurons following cortical microinjection in rats (11–14). Experimental evidence and computational network analyses using fluorescent calcium imaging have already demonstrated that bidirectional μTENNs exhibit functional connectivity and the potential for directed information transfer by at least 10 days in vitro (DIV) (15). Here, we advance the development of μTENNs into a putative “living electrode,” that is, a self-contained, implantable, axon- and synapse-based conduit for optically controlled neuronal activity and information transfer to/from the brain. In this approach, the μTENN is implanted at a predetermined depth to form synapses with local neural circuitry and propagate information along μTENN axons to/from an externalized apparatus at the brain surface (Fig. 1, I and J). Transduction of the μTENN neurons to express optogenetic proteins before implant would thus enable light-driven neuromodulation (through photostimulation of the μTENN neurons to influence downstream cortical activity) or monitoring (by recording μTENN neurons as a representation of multiple cortical synaptic inputs) (Fig. 1, I and J).

This living electrode approach as described may address multiple functional challenges in current neuromodulation and neural recording strategies. As engineered neural microtissue, living electrodes provide a natural biological substrate for signal transfer between the neural target and the brain surface through synapse formation with host neurons. Further, the wholly organic μTENN may ameliorate the chronic foreign body response as any inorganic materials (e.g., photodiode arrays) are relegated to the brain surface, potentially improving long-term stability (Fig. 1J). Combined with the targeting specificity of optogenetic neuromodulation, light-driven living electrodes could provide access to deep neuronal circuitry without the light penetration limitations or potential viral delivery risks of existing optogenetic approaches (as viral transduction is restricted to μTENN neurons before implant).

Motivated by these potential benefits, the present work details the biofabrication and characterization of a novel optically controlled living electrode approach to neural interface. Here, we present the following advancements in μTENN technology as a medium for neural interface: (i) the fabrication of next-generation, neuronal aggregate-based unidirectional (single-aggregate) and bidirectional (dual-aggregate) μTENNs; (ii) characterization of their growth and viability across multiple construct parameters; (iii) validation of their network formation and maturation over time; (iv) optical control and monitoring of optogenetically active μTENNs in vitro; (v) implantation and optical readout of aggregate-based μTENNs in rodent cortex as proof of concept for living electrodes; and (vi) histological evidence of a modest gliotic response to μTENNs, as well as evidence of neuronal survival, neurite outgrowth, and synaptogenesis with host. Through the successful creation and validation of implantable, optically controlled, and functional neuron- and axon-based microtissue, these developments represent a notable milestone toward the long-term goal of achieving a biologically based neural interface that offers improved selectivity and longevity compared to alternative nonorganic approaches.

MATERIALS AND METHODS

All procedures were approved by the Institutional Animal Care and Use Committees at the University of Pennsylvania and the Michael J. Crescenz Veterans Affairs Medical Center and adhered to the guidelines set forth in the National Institutes of Health Public Health Service Policy on Humane Care and Use of Laboratory Animals (2015).

Cortical neuron isolation and culture

Neural cell isolation and culture protocols are similar to that of published work (11, 12, 16). Briefly, timed-pregnant rats were euthanized, and the uterus was removed. Embryonic day 18 fetuses were transferred from the uterus to cold Hanks’ balanced salt solution (HBSS), wherein the brains were extracted and the cerebral cortical hemispheres were isolated under a stereoscope via microdissection. Cortical tissue was dissociated in 0.25% trypsin + 1 mM EDTA at 37°C for 10 to 12 min, after which the trypsin/EDTA was removed and replaced with deoxyribonuclease (DNase) (0.15 mg/ml) in HBSS. Dissociated tissue + DNase were centrifuged for 3 min at 3000 rpm before the DNase was removed and the cells were resuspended in serum-free neuronal culture media, composed of Neurobasal + B27 + Glutamax (Thermo Fisher Scientific) and 1% penicillin-streptomycin.

μTENN fabrication

μTENNs were constructed in a three-phase process (Fig. 1, A to C). First, agarose microcolumns of a specified geometry [outer diameter (OD), inner diameter (ID), and length] were formed in a custom-designed acrylic mold as described in earlier work (Fig. 1A) (13). The mold is an array of cylindrical channels that allow for the insertion of acupuncture needles (Seirin, Weymouth, MA) to form a void with the annular dimensions (OD × ID) of the microcolumn once assembled. The mold has been fabricated with more precise machining equipment relative to earlier work to improve coaxial alignment of the needles and channels. Molten agarose in Dulbecco’s phosphate-buffered saline (DPBS) was poured into the mold-needle assembly and allowed to cool (agarose, 3% weight/volume). Once the agarose solidified, the needles were removed and the mold was disassembled, yielding hollow agarose microcolumns with a specific OD equal to the size of the channels and ID equal to the OD of the needles. Microcolumns were sterilized via ultraviolet light for 30 min and stored in DPBS to prevent dehydration until needed. For these studies, the mold channels were 398 μm in diameter and the acupuncture needles were 180 μm, resulting in microcolumns with a 398-μm OD and a 180-μm ID. Microcolumns were cut to the desired length for each cohort as described below.

Next, primary cortical neurons were forced into spheroidal aggregates (Fig. 1C). Early-generation μTENNs were created by seeding microcolumns with dissociated primary cortical neurons, resulting in random clustering and multidirectional axonal growth (Fig. 1, D and E). The formation of neuronal aggregates before seeding provides more precise control over μTENN architecture, enabling the growth of long axonal fascicles spanning the length of the microcolumn (Fig. 1, F to H). To create aggregates, dissociated cortical neurons were suspended at a density of 1.0 million to 2.0 million cells/ml and transferred to an array of inverted pyramidal wells made in polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) cast from a custom-designed, three-dimensional (3D) printed mold (Fig. 1B). Neuron suspensions were then centrifuged in the wells at 200g for 5 min before being incubated overnight at 37°C, 5% CO₂. This centrifugation resulted in forced aggregation of neurons with precise control of the number of neurons per aggregate/sphere (12 μl of cell suspension per well). Pyramidal wells and forced aggregation protocols were adapted from Ungrin et al. (17).

Last, microcolumns were removed from DPBS and excess DPBS were removed from the microcolumn channels via micropipette. Microcolumns were then filled with extracellular matrix (ECM) composed of rat tail collagen (1.0 mg/ml) + mouse laminin (1.0 mg/ml) (Reagent Proteins, San Diego, CA) (Fig. 1C). Unidirectional or bidirectional μTENNs were seeded by carefully placing an aggregate at one (unidirectional) or both (bidirectional) ends of a microcolumn using fine forceps under a stereoscope and confirming aggregate adherence for 45 min at 37°C, 5% CO₂. Early-generation dissociated μTENNs were fabricated by transferring dissociated cortical neurons into the ECM-filled microcolumn via micropipette as detailed in prior work (11, 12). All μTENNs were grown in neuronal culture media with fresh media replacements every 2 DIV.

Growth characterization

Several groups of aggregate-based μTENNs were fabricated to assess growth across construct polarities (unidirectional or bidirectional) and lengths (from 2 to 9 mm). In addition, early-generation dissociated μTENNs were generated to compare growth of aggregate-based μTENNs to those generated using that previous methodology. In total, eight experimental groups were generated: dissociated/2 mm long (LE_DISS,2) (n = 7), unidirectional/2 mm long (LE_UNI,2) (n = 6), unidirectional/5 mm long (LE_UNI,5) (n = 3), bidirectional/2 mm long (LE_BI,2) (n = 15), bidirectional/3 mm long (LE_BI,3) (n = 12), bidirectional/5 mm long (LE_BI,5) (n = 17), bidirectional/7 mm long (LE_BI,7) (n = 8), and bidirectional/9 mm long (LE_BI,9) (n = 3). Low sample size for LE_BI,9was due to cessation of neurite outgrowth before 10 DIV without network formation (i.e., not forming interaggregate connections).

Phase-contrast microscopy images of μTENNs in culture were taken at 1, 3, 5, 8, and 10 DIV at ×10 magnification using a Nikon Eclipse Ti-S microscope, paired with a QIClick camera and NIS Elements BR 4.13.00. Growth rates for each group at specific time points were quantified as the change in the length of the longest identifiable neurite divided by the number of days between the current and preceding time point. The longest neurites were manually identified within each phase image using ImageJ (National Institutes of Health, MD), and length was measured from the edge of the source aggregate to the neurite tip. To standardize measurements, the edge of the source aggregate identified at 1 DIV was used as the reference point across subsequent time points. Growth was measured until axons crossed the length of the column (for unidirectional constructs) or axons crossed the distance between aggregates (for bidirectional constructs). Growth rates were averaged for each time point, with the average maximum and minimum growth rates and average crossing time compared across aggregate-based μTENNs with one-way analysis of variance (ANOVA). Post hoc analyses were performed where necessary with Bonferroni-corrected pairwise comparisons. For reference, planar cultures of cortical neurons (n = 10) were grown in parallel with μTENN cultures, with the longest identifiable neurites measured at 1, 3, and 5 DIV. Single neurites could not be identified at later time points due to culture maturation. Axonal outgrowth in planar cultures was taken as the average growth rate across time points, which were compared via unpaired t test.

To identify aggregate-specific growth across the microcolumns, cortical neuronal aggregates were labeled with either green fluorescent protein (GFP) or the red fluorescent protein mCherry via adeno-associated virus 1 (AAV1) transduction (Penn Vector Core, Philadelphia, PA). Briefly, after centrifuging aggregates in the pyramid wells, 1 μl of AAV1 packaged with the human synapsin-1 promoter was added to the aggregate wells (final titer, ~3 × 10¹⁰ genomic copies/ml). Aggregates were incubated at 37°C, 5% CO₂ overnight before the medium was replaced twice, after which transduced aggregates were plated in microcolumns as described above, each with one GFP⁺ and one mCherry⁺ aggregate (n = 6). Over multiple DIV, images of the μTENNs were taken on a Nikon A1RSI laser scanning confocal microscope paired with NIS Elements AR 4.50.00. Sequential slices of 10 to 20 μm in the z plane were acquired for each fluorescent channel. All confocal images presented are maximum intensity projections of the confocal z-slices.

Viability assessment

To assess neuronal viability in both newly formed and mature networks, unidirectional/5 mm (LE_UNI) and bidirectional/5 mm (LE_BI) μTENNs were grown to 10 (immature) and 28 (mature) DIV before staining with a calcein-AM/ethidium homodimer-1 (EthD-1) assay (Thermo Fisher Scientific). Metabolically active cells convert the membrane-permeable calcein-AM to calcein, which fluoresces green (λ_exc, ~495 nm; λ_em, ~515 nm), while EthD-1 enters membrane-compromised cells and fluoresces red upon binding to nucleic acids (λ_exc, ~495 nm; λ_em, ~635 nm). Age-matched, 2D cortical cultures were plated on polystyrene and stained to identify differences in survival due to the aggregate culture method.

Briefly, cultures were gently rinsed in DPBS at the selected time points. A solution of calcein-AM (1:2000 dilution; final concentration of ~2 μM) and ethidium homodimer-1 (1:500; ~4 μM) in DPBS was added to each culture, followed by incubation at 37°C, 5% CO₂ for 30 min. Following incubation, cultures were rinsed twice in fresh DPBS and imaged at ×10 magnification on a Nikon A1RSI laser scanning confocal microscope paired with NIS Elements AR 4.50.00. Viability was quantified as the ratio of the total area of calcein-AM⁺ cells to the total area of both calcein-AM⁺ and ethidium-homodimer⁺ cells using ImageJ (National Institutes of Health, MD). Sample sizes for each group were as follows: LE_UNI,5mm (n = 4, 4); LE_BI,5mm (n = 7, 4); planar cultures (n = 9, 5) for 10 and 28 DIV, respectively.

Optical stimulation, calcium imaging, and optical recording analysis

As proof-of-concept validation for the optically controlled living electrode approach, we established an “all-optical” paradigm enabling the simultaneous optogenetic control and optical monitoring of aggregate-based μTENNs in vitro. Cortical neuronal aggregates were transduced with either channelrhodopsin-2 (ChR2) for light-based neuronal activation (“input”) or the genetically encoded fluorescent calcium reporter RCaMP1b for optical readout of neuronal activity (“output”) via AAV1 transduction as described above (Penn Vector Core). Five- to 6-mm-long bidirectional μTENNs were then plated with one input/ChR2⁺ aggregate and one output/RCaMP1b⁺ aggregate (n = 5). ChR2 and RCaMP have been investigated and used for all-optical electrophysiology in vitro with minimal spectral overlap, reducing the likelihood of false-positive responses in RCaMP⁺ neurons due to photostimulation of ChR2⁺ neurons (18). At 10 DIV, μTENNs were stimulated via an LED optical fiber positioned approximately 1 to 3 mm above the input aggregate, such that the entire aggregate was illuminated. A Plexon Optogenetic Stimulation System with LED modules for each desired wavelength was used to stimulate the μTENNs (Plexon Inc.). Stimulation consisted of a train of 10 100-ms pulses (1 Hz) at 465 nm, within the excitation spectra of ChR2. Each train was repeated three times for a given LED current amplitude (50, 100, 200, 250, 300 mA); amplitudes corresponded to approximate stimulation intensities of 106, 211, 423, 528, and 634 mW/mm² from the tip of the optical fiber and 4.7, 9.3, 18.7, 23.3, and 28.0 mW/mm² at the aggregate, respectively. As a control, μTENNs were stimulated as above at 620 nm (outside of the excitation spectra of ChR2) at 300 mA/28.0 mW/mm². Recordings of the μTENNs’ output aggregates were acquired at 25 to 30 frames per second on a Nikon Eclipse Ti microscope paired with an ANDOR Neo/Zyla camera and Nikon Elements AR 4.50.00 (Nikon Instruments).

Following optical stimulation and/or recording, calcium imaging acquisitions were manually reviewed against phase images of the same μTENNs to identify regions of interest (ROIs) containing neurons and background ROIs. Neuronal ROIs were identified as areas clearly containing one or more neuronal cell bodies that exhibited repeated synchronous changes in pixel intensity, although because of the dense packing of the aggregate, these ROIs also contained axons. Background ROIs were empty square areas containing no neurons (i.e., only culture media). The mean pixel intensities for each ROI were imported into MATLAB for further analysis via custom scripts (MathWorks Inc.). Within MATLAB, the background ROI intensity for each recording was subtracted from active ROIs. Ten such ROIs were randomly selected and averaged to obtain a representative fluorescence intensity trace across each output aggregate. Subsequently, the percent change in fluorescence intensity over time (∆F/F_o) was calculated for each mean ROI, where ∆F equals (F_T − F_o), F_T is the mean ROI fluorescent intensity at time T, and F_o is the average of the lower half of the preceding intensity values within a predetermined sampling window (19). The peak ∆F/F_o for each train was averaged per μTENN for each of the given stimulation intensities. The average maximum ∆F/F_o was then compared across stimulation intensities with a one-way ANOVA, with post hoc analysis performed where necessary with the Tukey procedure. In addition, the peak ∆F/F_o of the output aggregate under 620-nm stimulation was used as a control (620 nm being outside of ChR2’s activation spectra) and compared to that under 465-nm stimulation at 300 mA/28 mW/mm² using an unpaired t test.

Immunocytochemistry

To determine whether aggregate-based μTENNs matured and attained the desired network architecture over time, μTENNs were grown and fixed in 4% formaldehyde for 35 min at 4, 10, and 28 DIV (n = 6, 4, and 8, respectively). μTENNs were then rinsed in 1× PBS and permeabilized with 0.3% Triton X-100 + 4% horse serum in PBS for 60 min before being incubated with primary antibodies overnight at 4°C. Primary antibodies were Tuj-1/β-tubulin III (1:500; T8578, Sigma-Aldrich) to label axons and synapsin-1 (1:500; A6442, Invitrogen) to label presynaptic specializations. Following primary antibody incubation, μTENNs were rinsed in PBS and incubated with fluorescently labeled secondary antibodies (1:500; sourced from Life Technologies and Invitrogen) for 2 hours at 18° to 24°C. Last, Hoechst (1:10,000; 33342, Thermo Fisher Scientific) was added for 10 min at 18° to 24°C before rinsing in PBS. μTENNs were imaged on a Nikon A1RSI laser scanning confocal microscope paired with NIS Elements AR 4.50.00. Sequential slices of 10 to 20 μm in the z plane were acquired for each fluorescent channel. All confocal images presented are maximum intensity projections of the confocal z-slices.

Cortical implantation and intravital calcium imaging

As proof-of-concept validation for optical living electrodes in vivo, bidirectional, approximately 1.5-mm-long (n= 6) or 5.5-mm-long (n = 4) μTENNs expressing GCaMP were delivered into the brain via stereotaxic microinjection using similar methodology to that described in prior work (11, 12). Male Sprague-Dawley rats weighing 325 to 350 g were anesthetized with isoflurane at 1.0 to 2.0 liters/min (induction, 5.0%; maintenance, 1.5-2.0%) and mounted in a stereotactic frame. Meloxicam (2.0 mg/kg) and bupivacaine (2.0 mg/kg) were given subcutaneously at the base of the neck and along the incision line, respectively. The area was shaved and cleaned with betadine solution, after which a small craniotomy was made over the primary visual cortex (V1) [coordinates: −5.0-mm anterior-posterior (AP), ±4.0-mm medial-lateral (ML) relative to bregma]. μTENNs were carefully loaded into a needle under a dissecting microscope by using fine forceps to gently manipulate the microcolumn into the needle shaft. The needle containing the μTENN was then coupled to a Hamilton syringe and mounted onto a stereotactic arm. To deliver the constructs into the brain without forcible expulsion, the needle was mounted on a micromanipulator and slowly inserted into the cortex to a depth of 1.0 mm such that the dorsal μTENN terminal was left ~500 μm above the brain surface. The plunger of the Hamilton syringe was then immobilized while the needle containing the μTENN was slowly raised. This method effectively deposited the μTENN in the wake of the needle withdrawal to minimize forces on the preformed neural network. Upon needle removal from the brain, the dorsal aggregate of the μTENN was immersed in artificial cerebrospinal fluid warmed to 37°C. To protect the dorsal μTENN terminal and enable imaging of the μTENN and surrounding tissue, two custom-made PDMS rings (OD, 5.0 mm; ID, 2.0 mm; thickness, 0.35 mm) were placed over the craniotomy/μTENN and secured to the skull with cyanoacrylate glue. A 3.0-mm-diameter glass coverslip was sandwiched between the two rings.

To assess whether μTENN neurons could deliver optical readout following microinjection, animals were anesthetized and mounted on a stereotactic frame for multiphoton calcium imaging of the μTENN neurons following a recovery period, i.e., at 5 and 10 days after implant. μTENNs were imaged on a Nikon A1RMP+ multiphoton confocal microscope paired with NIS Elements AR 4.60.00 and a 16× immersion objective. Recordings of the μTENNs’ dorsal aggregates were taken at 3 to 5 frames per second, similarly to other intravital work (20). After recording, ROIs of μTENN neurons were manually identified, with the mean pixel intensity of each ROI plotted over time as an aggregate-level measure of neuronal activity. To distinguish neuronal activity from the animal breathing artifact, the fast Fourier transform (FFT) of the mean pixel intensity averaged across 10 to 15 ROIs was used to identify the frequency peak(s) associated with the observed breathing rate during imaging. Peaks were identified as frequencies whose amplitudes were 2 SDs or more than the average amplitude of the Fourier spectra.

Tissue harvest and histology

At 1 week and 1 month after implant, rats were anesthetized with euthasol (150 mg/kg) (Midwest) and transcardially perfused with cold heparinized saline and 10% formalin. After postfixation of the head overnight, the brain was harvested and stored in PBS to assess μTENN survival and host/μTENN synaptic integration (n = 10). Histology was performed via traditional immunohistology (IHC) and the Visikol clearing method to resolve thicker tissue sections where appropriate.

For traditional IHC, brains were blocked (sagittally for longitudinal μTENN visualization and obliquely for axial μTENN visualization) and cut in 20- or 40-μm slices for cryosectioning. For frozen sections, slices were air-dried for 30 min, twice treated with ethanol for 3 min, and rehydrated in PBS twice for 3 min. Sections were blocked with 5% normal horse serum (ABC Universal Kit, Vector Labs, catalog no. PK-6200) in 0.1% Triton X-100/PBS for 30 to 45 min. Primary antibodies were applied to the sections in 2% normal horse serum/Optimax buffer for 2 hours at room temperature. In longitudinal sections, primary antibodies labeled neurons/axons (1:1000; rabbit anti-NF200), neurons/dendrites (1:1000; chicken anti-MAP2), neurons/axons (1:1000; mouse anti-Tuj1), and/or presynaptic terminals (1:1000; mouse anti-synapsin). In axial sections, primary antibodies labeled microglia/macrophages (1:1000; rabbit anti–Iba-1) and astrocytes [1:1000; goat anti-glial fibrillary acidic protein (GFAP)]. Sections were rinsed with PBS three times for 5 min, after which secondary antibodies (1:1000) were applied in 2% normal horse serum/PBS for 1 hour at room temperature. Sections were counterstained with DNA-specific fluorescent Hoechst 33342 for 10 min and then rinsed with PBS. After immunostaining, slides were mounted on glass coverslips with Fluoromount-G mounting media.

In the Visikol method, brains were glued to a vibratome mounting block directly in front of a 5% low electroendoosmosis (EEO) agarose post (Sigma-Aldrich, A-6103) and placed in PBS surrounded by ice. The brain was cut in 100- to 200-μm coronal segments with a Leica VT-1000S vibratome until the μTENN implantation site was approximately 1 mm from the cutting face. Subsequently, a single 2-mm section containing the μTENN was cut and placed in PBS (frequency setting, 9; speed, 10). The 2-mm brain section was treated at 4°C with 50, 70, and 100% tert-butanol, each for 20 min. After the ascending tert-butanol steps, the tissue was removed and placed on a Kimwipe to carefully remove any excess reagent. Visikol HISTO-1 was applied to the sample for 2 hours at 4°C followed by Visikol HISTO-2 for 2 hours at 4°C to complete the clearing process. The sample was placed in a petri dish, and a hydrophobic well was drawn around the tissue. Fresh Visikol HISTO-2 was applied to completely submerge the tissue, which was then covered by a glass coverslip.

Coverslips containing brain slices were imaged on a Nikon A1RMP+ multiphoton confocal microscope paired with NIS Elements AR 4.60.00 and a 16× immersion objective. A 960-nm laser was used to visualize the μTENNs containing neurons expressing GFP/GCaMP for qualitative observations of neuronal presence, location, and construct architecture after implant.

General statistical methodology

The Shapiro-Wilk test was used to test data for normality before statistical comparisons. Unless otherwise specified, ANOVA was used with post hoc analyses as appropriate. The threshold for statistical significance was defined in all cases as P < 0.05. All data are presented as means ± SEM.

RESULTS

The objectives of our current efforts were threefold: (i) to reproducibly fabricate living electrode aggregate–based μTENNs and characterize their growth, viability, and network architecture; (ii) to demonstrate the ability to control and monitor μTENNs via light; and (iii) to determine whether living electrode neurons survive in vivo and remain viable for optical monitoring once transplanted in the host cortex.

Fabrication and axonal outgrowth

A fundamental step in the creation of μTENN-based living electrodes is a fabrication method that enables control and consistency of their structure across preparations. In earlier work, μTENNs were seeded with dissociated cortical neurons suspended in growth media, which, in many cases, formed clusters at random throughout the microcolumn interior (Fig. 1, D and E). By instead inducing the neurons to form spheroidal aggregates before their plating, these newly developed aggregate-based μTENNs consistently generated the desired cytoarchitecture of discrete somatic and axonal zones in vitro (Fig. 1, F to H). This macro-level aggregate-based μTENN structure was demonstrably reproducible across both unidirectional and bidirectional polarities and a range of microcolumn parameters (e.g., 2 to 9 mm in length), although, as described below, growth dynamics varied across these design parameters.

In practice, implanted μTENNs may need to integrate with targets several millimeters below the brain surface; hence, a critical milestone was the establishment of long axons in a new 3D microenvironment, as well as early characterization of these axonal growth dynamics to begin exploring the practical constraints of the aggregate culture method. Qualitatively, phase microscopy revealed healthy and rapid axonal outgrowth across all aggregate-based μTENNs through the ECM core; observed growth rates generally peaked at 3 to 5 DIV before slowing as unidirectional μTENN axons reached the opposing end (Fig. 2A) or bidirectional μTENN axons from opposing aggregates grew toward and along each other (Fig. 2, B and C). For both μTENN polarities, the maximal growth rate increased with the construct length (Fig. 2D), with one-way ANOVA of the average maximum growth rate implicating the LE cohort as a significant effect (F-statistic = 14.1, P < 0.0001). The fastest growth rate was observed in bidirectional 9-mm (LE_BI,9) μTENNs, which reached 1101.8 ± 81.1 μm/day at 3 DIV—nearly 17× the rate of early-generation μTENNs (Fig. 2, D and F, and table S1).

Since the cohort was a statistically significant factor, subsequent Bonferroni-corrected pairwise comparisons showed that the average maximum growth rates of the longest cohorts (LE_{BI, 5}, LE_BI,7, and LE_BI,9) were indeed statistically higher than those of the shortest cohorts LE_BI,2 and LE_BI,3 (P < 0.001), while only LE_BI,9 surpassed the unidirectional cohorts with significance (LE_UNI,2: P < 0.01; LE_UNI,5: P < 0.05) (Fig. 2D). In addition, both dissociated μTENNs and control planar neuronal cultures exhibited similar growth rates that were an order of magnitude slower than their aggregate counterparts; peak growth rates were 61.7 ± 5.01 (LE_DISS) and 39.1 ± 20.6 (CTRL) μm/day, respectively (Fig. 2F and table S1).

While the longest μTENNs exhibited the fastest peak growth rates, they took the most time to cross the microcolumn length. In addition, several constructs in the LE_BI,7 and LE_BI,9 cohorts exhibited a decline and eventual cessation of measurable outgrowth before fully forming networks. Similarly, one-way ANOVA and Bonferroni post hoc analysis of the average crossing time showed that LE_BI,7 and LE_BI,9 axons crossed the length of the microcolumn significantly later than those of LE_UNI,2, LE_BI,2, LE_BI,3, and LE_BI,5 (F-statistic = 12.99, P < 0.0001 to 0.05) (Fig. 2E). LE_UNI,5 axons did not, on average, fully cross the construct length by 10 DIV (Fig. 2F and table S1). Notably, one-way ANOVA of the average minimum growth rate did not detect any significant differences across aggregate-based LE groups (F-statistic = 1.17, P = 0.332).

μTENN viability

Neuronal maturation and network development are denoted by a complex interplay of growth, pruning (elimination), and remodeling of synaptic connections as individual neurons form larger networks (21). For μTENNs, phase/fluorescent images and calcium imaging analyses of bidirectional aggregate-based μTENNs provide structural and functional evidence for the presence of an initial interaggregate network by 10 DIV (15). To assess the impact of network maturation and pruning on neuronal survival in these constructs, unidirectional and bidirectional μTENNs were grown to 10 and 28 DIV. Survival was quantified as the ratio of the summed area of calcein-AM⁺ (live) cells to that of all (i.e., both calcein-AM⁺ and ethidium homodimer⁺) cells at the specified time points, with age-matched planar cultures on polystyrene as controls for the aggregate culture method (fig. S1, A to F). μTENN neurons survived up to at least 28 DIV, with qualitative observation of survival out to 40 DIV (fig. S1I). Although ANOVA identified the DIV as a significant main effect (F-statistic = 32.21, P < 0.0001), the LE/culture group was not a significant factor (P > 0.84). The interaction effect was significant (P < 0.01), so Bonferroni analysis was used to compare groups at each time point (fig. S1G). Survival of planar cultures at 28 DIV (53.6%) was found to be statistically lower than that of LE_UNI(80.3%) (P < 0.05), LE_BI (84.8%) (P < 0.001), and planar cultures (97.7%) (P < 0.0001) at 10 DIV (fig. S1G). Moreover, planar culture viability at 10 DIV surpassed those of both LE_UNI (68.1%) and LE_BI (69.0%) at 28 DIV (P < 0.01). Overall, planar cultures exhibited an average 45% reduction in viability from 10 to 28 DIV, while LE_UNI and LE_BI showed a 15.2 and 18.6% drop over time, respectively (fig. S1H).

μTENN architecture and synaptogenesis

To characterize changes in μTENN architecture over time, bidirectional μTENNs were engineered to express GFP and mCherry to track cross-aggregate neurite outgrowth and integration. μTENNs were fixed and immunolabeled to determine the spatial distribution of cell somata/nuclei, axons, and synapses at set time points before and during network formation (Fig. 3). Transducing each aggregate before seeding enabled the consistent identification of aggregate-specific projections over time (Fig. 3A), even within dense axonal bundles (Fig. 3, B to D). Confocal images of GFP/mCherry μTENNs revealed that projections from each aggregate made contact and grew along opposing axons toward the opposite aggregate (Fig. 3, E to G); qualitatively, no gross trends were observed in the extent of physical interaction and integration between the two neuronal populations labeled with either reporter. Immunolabeling of cell nuclei with Hoechst revealed that neuronal somata remained localized almost exclusively to the aggregates across time points, which were spanned by long bundles of Tuj-1 expressing axonal projections through the microcolumn lumen (Fig. 3H). Synapsin-1 is expressed at the presynaptic terminals of mature neurons and is involved in the regulation of myriad developmental processes, including the formation and maintenance of synapses—making it a suitable proxy for characterizing the neuronal maturity in culture (22–25). Immunolabeling revealed highly dense intra-aggregate clusters of axons and synapses within μTENNs at all time points, presumably formed upon or shortly after plating (Fig. 3, I and J). No statistically significant differences in synapsin distribution were identified; however, qualitative observations of synapsin-1 expression showed a moderate increase in synapsin-1⁺ puncta within the μTENN aggregates as well as along the axonal tracts within the microcolumn lumen over time (fig. S2). Immunolabeling thus suggests that neurons within bidirectional μTENNs reach a mature state, i.e., are capable of both intra-aggregate and interaggregate synaptogenesis following axonal growth.

Calcium imaging and optical stimulation

Bidirectional μTENNs expressing the calcium reporter GCaMP6f exhibited spontaneous oscillations at and below the delta band (0.5 to 5 Hz) in the absence of external stimulation (Fig. 4, A and B; see movies S1 to S3). These observations reflect earlier findings wherein GCaMP⁺ μTENNs demonstrated functional connectivity with strongly correlated oscillations in the delta and theta bands, or 1 to 8 Hz, across aggregates (15). The abundance of synapsin expression coupled with synchronicity of oscillation between aggregates further support the conclusion that aggregate-based μTENNs form functional synaptic networks with a coherent structure that may be observed at both the neuron and aggregate levels.

Bidirectional μTENNs were also engineered to enable light-based stimulation and concurrent calcium imaging in vitro by transducing one aggregate with ChR2 and the opposing aggregate with RCaMP (Fig. 4, C and D). Upon illumination of ChR2⁺ (input) aggregates with 465-nm light (within the excitation spectrum of ChR2), the opposing RCaMP⁺ (output) aggregates exhibited time-locked changes in fluorescence intensity in response (Fig. 4, E to H). All-optical μTENNs were fabricated 5 mm in length with wavelength-separated vectors to reduce the probability of photostimulation artifact. To further reduce the potential for confound from photostimulation artifact, constructs’ input aggregates were illuminated with 620-nm light (outside of the ChR2 activation spectrum) of equal intensity to serve as a negative stimulation control. Photostimulation at 620 nm induced no readily observable responses; the mean peak ∆F/F_o of the output aggregate was significantly greater under 465-nm stimulation than 620-nm stimulation at 28.0 mW/mm² (P < 0.05) (Fig. 4I). Collectively, these findings suggest that the changes in ∆F/F_o under 465-nm stimulation reflected synaptically mediated firing of neurons in the output aggregate in response to light-based activation of neurons within the input aggregate. Although there was high variability in ∆F/F_o between μTENNs, the percent change in the fluorescence of the output aggregate relative to baseline under optical stimulation could be reproducibly distinguished from endogenous activity across all the μTENNs studied; further, the average maximum ∆F/F_opositively correlated with the stimulation intensity (Fig. 4I). Overall, these results suggest that photostimulation of the input aggregates resulted in controllable signal propagation and modulation of activity in the associated output aggregates.

Implantation and intravital calcium imaging

Following in vitro characterization, 1.0- to 1.2-mm aggregate-based μTENNs were fabricated as described above, transduced to express GCaMP6, and implanted in rodent V1 via stereotactic microinjection as a proof of concept for living electrode survival and function in vivo. Following μTENN delivery, a cranial window was affixed to a custom-build PDMS ring assembly adhered to the surrounding skull to permit repeated noninvasive monitoring of the dorsal μTENN aggregate and surrounding V1 (Fig. 5A). This noninvasive imaging was performed using multiphoton microscopy in animals anesthetized using controlled isoflurane delivery with carefully monitored, voluntary respiration. Acute postoperative fluorescent imaging confirmed that the bulk of the dorsal aggregate remained intact at the original implant location (Fig. 5, B and C). Multiphoton calcium imaging of the dorsal aggregate was performed at 5 and 10 days to avoid potential confounds from the microtrauma of the immediate posttransplant environment affecting neuronal activity. This calcium imaging revealed surviving and active GCaMP⁺ μTENN neurons in vivo, with transients in GCaMP intensity in the delta band (1 to 5 Hz) (Fig. 5, D to F). Putative activity was also present at frequencies below 1 Hz (Fig. 5F, inset). Of note, the respiration of the animals caused a consistent artifact in the recorded fluorescence signals resembling a low-frequency oscillation in the baseline fluorescence across all ROIs; however, this respiratory artifact could be readily identified and isolated within the FFT of the time-lapse recordings as a ~0.5- to 0.7-Hz peak distinct from both the delta band and putative sub–1 Hz activity (red band, Fig. 5F). The intravital μTENN neuronal activity patterns were similar to those recorded from age-matched nonimplanted μTENNs in vitro, suggesting that aggregate-based μTENNs retain their network structure over days after transplant. Longer-term studies will evaluate the utility of μTENNs as an output for neuronal activity as host neurons integrate with the ventral terminus of the network, which would presumably alter the network dynamics.

At 1 week and 1 month after injection in the rodent brain, immunohistochemistry in optically cleared as well as sectioned tissue revealed that μTENN neurons survived and maintained the preformed somatic-axonal architecture, with GFP⁺/GCaMP⁺ cell bodies predominantly localized to the microcolumn terminals and spanned by axonal tracts (Fig. 6). Staining for markers of astrogliosis and microgliosis in axial sections revealed only a modest host inflammatory reaction to the μTENNs (Fig. 6C). Large, dense clusters of GCaMP⁺cell bodies were found at the dorsal regions of implantation (Fig. 6B), with axons and dendrites within the lumen spanning the microcolumn (Fig. 6D). Dorsal aggregates were observed spreading along the brain surface or further along the microcolumn, putatively due to a cell migration away from the aggregate to increase interactions with host tissue (Fig. 6B). In some cases, there was also neuronal migration up to several millimeters from the ventral implant location, although the presence and extent of migration varied across implants. In general, there was widespread neurite outgrowth from the ventral aggregate of the living electrode, with structural evidence of synapse formation with host neurons (Fig. 6, E and F).

DISCUSSION

Microelectrodes—the current gold standard for recordings—have functioned in neural interfaces successfully on the order of months and, less frequently, years, in rodents, nonhuman primates, and human patients (1, 26–28). However, microelectrode-based BCIs generally succumb to a complex combination of abiotic and biological factors (including host neuronal loss, gliosis, biofouling, electrode movement, and/or mechanical failure) that impede stability, specificity, and clinical deployment (1–5, 29). Optogenetic strategies for neuromodulation enable more selective stimulation but must restrict the vector of interest to targeted cells, address the scattering and limited tissue penetration of light, and activate transduced cells without overheating brain tissue (30–33). Ongoing efforts to minimize inflammation have yielded more compliant electrodes and electrode coatings/cofactors; however, the chronic foreign body response, consequent signal drop, and increase in stimulation thresholds continue to negatively affect current neural interface systems.

μTENNs as living electrodes may present an alternative to conventional microelectrode- or optogenetic-based strategies for neuromodulation/recording with improved biocompatibility, selectivity, and longevity. The integration of living cells as functional device elements is not a new concept, for instance, other groups have exploited electroactive bacteria for biosensing applications (34). Similar efforts to develop biohybrid electronics include the introduction of living neural progenitors (or neural-like PC12 cells) and/or glial support cells into biocompatible hydrogels or hydrogel-polymer composites; these “biosynthetic hydrogels” are then layered to coat metallic electrodes (35–37). However, the living electrode approach presented in this work is distinct as an implantable, anatomically inspired, 3D engineered microtissue preformed in vitro before delivery into the brain. This approach uses aggregate-based μTENNs, which are designed to form a relay for biological (i.e., synaptic) interface with existing neuronal circuitry while remaining accessible at the brain surface for optical stimulation (input) or imaging (output). By enabling the segregation of cell bodies (aggregates) and axons in a single structure, these μTENNs may be considered a physical abstraction of the mammalian neural connectome, wherein locally connected neuronal populations are spanned by long distance axonal pathways.

The work presented here sets a critical foundation in developing aggregate-based μTENNs toward a viable neural interface and provides insight into the experimental milestones needed to scale more long-term translational challenges for similar “living” interfaces. In addition, these constructs consistently generate the connectome architecture described above based on the few design parameters (length and polarity) explored in this work. In reality, the biofabrication method developed for aggregate-based μTENNs provides several degrees of control over their microenvironment through systematic variation of its material (e.g., microcolumn curvature, stiffness, and bioactivity), chemical (ECM composition and growth factor gradients), and cellular [phenotype(s) and expression] properties. There are thus unexplored opportunities for aggregate-based μTENNs to serve as a multifaceted and scalable investigative tool both in vitro and in vivo for the neuroscience field.

Early growth characterization demonstrated that these next-generation μTENNs, created using forced neuronal aggregation and improved microfabrication techniques, can grow to at least 9 mm while maintaining the desired cytoarchitecture. Further, they exhibit accelerated axonal outgrowth and greater longitudinal “bundling” of axons than in early-generation dissociated μTENNs (Fig. 1, E and H). The observed growth rates for axons in dissociated μTENNs averaged ~60 μm/day over the first 3 days, reflecting growth trends in literature for cortical axons (38, 39). In comparison, the peak axonal growth rates from aggregate-based μTENNs exceeded the rate exhibited by dissociated neurons by two orders of magnitude, with the LE_BI,9 group attaining the remarkable axon extension rate of more than 1000 μm/day (Fig. 2). Neuronal aggregation likely induced bundled and directed neurite extension and may positively influence axon-ECM interactions with the collagen and laminin comprising the microcolumn lumen. Other potential factors for such accelerated axonal outgrowth from the neuronal aggregates include the restriction of axonal growth to the microcolumn interior and the lack of synaptic targets within the microcolumn, which may reduce nonlinear axon branching between aggregates and permit more efficient growth cone migration within the lumen (38, 40–43). Although peak growth rates across aggregate-based μTENN groups were positively correlated with μTENN length, their initial growth rates did not vary significantly. This may implicate a critical distance between aggregates below which neuronal processes meet before reaching their maximum growth rates. This distance may be required for the neurons to establish sufficient neurotrophic support and/or up-regulate the growth machinery required to reach these speeds.

With respect to neuronal health in aggregates, neuronal attrition from 10 to 28 DIV was ~2.4 to 3× lower in unidirectional and bidirectional μTENNs than in control planar cultures (fig. S1, G to H). Established work has shown that neurons exhibit better growth and survival in 3D environments, which more accurately approximate conditions in vivo (44). Similarly, the connectome-inspired 3D microstructure of spheroidal aggregates and axonal bundles appears to provide cortical neurons with the requisite conditions for rapid axonal growth and prolonged viability. μTENNs also demonstrated survival out to at least 40 DIV (fig. S1I), enabling more chronic in vitro investigations of these dynamics and their mechanisms. Potential subsequent steps include the measuring of neuron health and functionality over time across various cell preparations, neuronal phenotypes, and alternative design parameters.

Beyond axonal growth and survival, another notable outcome was the structural and functional connectivity between aggregates for bidirectional μTENNs. Our biofabrication methodology enabled for aggregate-specific transduction and labeling of individual μTENNs such that we could track structural integration between neuronal populations as the constructs matured (Fig. 3, A to G). Immunocytochemistry provided additional evidence of network maturation with the presence and proliferation of synapsin⁺ puncta from as early as 4 DIV to at least 28 DIV (fig. S2). As the primary points of contact and communication between neurons, synapses are often used to determine the functional maturity of neuronal networks (25, 45). These results suggest that μTENN neurons in aggregates begin to form synaptic networks soon after plating, which mature and expand over time, findings consistent with those in planar cortical cultures (25). Intra-aggregate synapses presumably dominate the synaptic population before axons span the aggregates to form interaggregate synapses. Future structural connectivity studies may build on the aggregate-specific labeling here to characterize intra- and interaggregate connectivity with greater resolution.

With respect to functional connectivity, we measured spontaneously generated calcium reporter activity in and across the GCaMP⁺ aggregates of bidirectional μTENNs (Fig. 4 and movies S1 and S2), which consisted predominantly of delta-band oscillations (1 to 5 Hz). High synchronicity across aggregates has been identified in delta-band oscillations through concurrent network analyses of 1.0- to 1.2-mm bidirectional GCaMP⁺ μTENNs in vitro, confirming their functional connectivity and capacity for information transfer (15). Within all-optical 5- to 6-mm μTENNs, neurons in the RCaMP⁺ aggregate for optical output displayed similar high-amplitude delta-band activity, with the addition of smaller transients corresponding to photostimulation delivered at the ChR2⁺ aggregate for light input (Fig. 4, G to I, and movie S3). The evoked activity was wavelength dependent, with a positive correlation to stimulus intensity that strongly suggested increased activation of ChR2⁺ neurons (Fig. 4I). These observations validated the presence of functional, long-distance axonal tracts and synaptic integration between two aggregate populations and, crucially, demonstrate the ability of these constructs to serve as an optically driven input-output platform for the transmission of information. Variability in the input-output response behavior may be attributed to the stimulation source size and position relative to the aggregate, which as a densely packed 3D sphere would inherently experience nonuniform illumination. Techniques for more uniform spheroidal photostimulation (e.g., with multiple light sources) or sufficient single-source stimulation to approximate the same (e.g., through depolarization of a critical fraction of ChR2⁺ neurons) remain an ongoing objective.

After transplant into rat cortex, we observed both delta-band oscillations (similar to those measured in vitro) and slow-wave oscillations below 1 Hz in GCaMP⁺ μTENN neurons (Fig. 5). Slow-wave oscillations have been recorded under anesthesia and during slow-wave sleep, as well as in cortical neuronal cultures in vitro (46, 47). Whether the detected <1 Hz of activity represents a response to cortical synaptic inputs remains beyond the scope of the current manuscript but seems unlikely, given the relatively acute imaging time points. However, the detection of calcium transients in μTENN neurons demonstrates they remain viable posttransplant, while sustained delta-band activity suggests active maintenance of the required network structure for synaptic transmission. A more direct assessment of functional integration over time would comprise longitudinal comparative measurements of intravital μTENN activity and host circuitry.

Histological observations at 1 week and 1 month revealed gross preservation of construct architecture and neurite outgrowth at the μTENN terminals (Fig. 6), reflecting similar findings with early-generation μTENNs in prior work. Further, colocalization of GCaMP⁺ processes with synapsin⁺ puncta suggests the structural formation of synapses with the cortex, although more functional validation of these histological implications in vivo is required as described above. The information transfer bandwidth of a living electrode-based interface is shaped by the extent of its synaptic integration with the brain. While this may enable highly dense, long-term stability as observed with native synaptic connections, controllability over the targeting of synaptic integration and neuronal migration remains a complex, nontrivial translational challenge that may be addressed through one or more strategies. Potential physical methods include a porous membrane at the ventral microcolumn terminal to restrict neuronal migration while permitting axonal projections between the μTENN and host brain. The μTENN itself may be fabricated with a neuronal phenotype known for its desired synaptogenetic behaviors or genetically engineered for more controllable synaptogenesis versus widespread outgrowth through the up-/down-regulation of specific developmental proteins (23, 48). Last, more direct regulation of the transplant environment may also promote more targeted integration, for instance, by introducing or promoting expression of trophic factors implicated in axonal guidance and/or synaptic pruning during development (21, 49). Another key concern for implantable interfaces is the invasiveness of delivery. Combined with well-established stereotactic neurosurgical techniques, the living electrode microcolumns may be further minimized to reduce the microinjection footprint. In addition, their material properties (e.g., stiffness) and potential cofactors (e.g., anti-inflammatory/growth factor cues) may be tailored against any subsequent foreign body response.

In summary, we have used optogenetic and tissue engineering techniques to create so-called living electrodes—cylindrical, hydrogel-encapsulated neuronal populations linked by functional axonal tracts—and demonstrated their biofabrication and functional validation in vitro, as well as their targeted delivery, survival, and continued function posttransplant. The results described indicate that aggregate-based μTENNs quickly and consistently form similar functional architecture—important for the biofabrication and scale-up of experimentally useful constructs—which is maintained over weeks in vitro. Thus, μTENNs may serve as an ideal system for studying neuronal growth, maturation and network dynamics due to their “abstraction” of mammalian brain connectome into individual units (44). While the constructs in this study were composed predominantly of glutamatergic neurons, μTENNs may be seeded with aggregates of different neuronal phenotypes or sources. Within the living electrode paradigm, this may enable the delivery of light-driven, synaptically transduced neuromodulation in vivo for specific clinical effects, for instance, neural circuit inhibition or diffuse neurotransmitter release. Multiple distinct μTENNs could also be grown in proximity to form connected assemblies, comprising a modular “building-block” approach to model, manipulate, and characterize systems-level network dynamics across neuronal circuits of mixed cell population and increased pathway complexity in vitro. Last, aggregate-based μTENNs were able to survive, grow, and provide GCaMP-based readout following implantation as proof-of-concept validation for preformed, implantable, biological pathways to synaptically integrate with, probe, and modulate neuronal targets through optical interface on the brain surface. These studies lay the groundwork for more in-depth investigations of the utility of aggregate-based μTENNs as both an experimental tool for in vitro modeling and a translational tool for neural interface following targeted transplant in the cerebral cortex or other anatomical targets.

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/7/4/eaay5347/DC1

View/request a protocol for this paper from Bio-protocol.https://creativecommons.org/licenses/by/4.0/

This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Acknowledgments: Funding: Financial support was primarily provided by the NIH [BRAIN Initiative U01-NS094340 (D.K.C.), T32-NS043126 (J.P.H.), and T32-NS091006 (L.A.S.)] and the NSF [Graduate Research Fellowship DGE-1321851 (D.O.A.)], with additional support from the Penn Medicine Neuroscience Center (D.K.C.), American Association of Neurological Surgeons and Congress of Neurological Surgeons [Codman Fellowship in Neurotrauma and Critical Care (D.P.)], and the Department of Veterans Affairs [Merit Review I01-BX003748 (D.K.C.), Merit Review I01-RX001097 (D.K.C.), Career Development Award #IK2-RX001479 (J.A.W.), and Career Development Award #IK2-RX002013 (H.I.C.)]. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the NIH, NSF, or Department of Veterans Affairs. Author contributions: Conceptualization: D.K.C., J.A.W., M.D.S., and H.I.C. Methodology: D.K.C., D.O.A., L.A.S., J.P.H., A.D.N., J.C.B., D.P., H.I.C., and J.A.W. Formal analysis: D.O.A. Investigation: D.O.A. and J.C.B. Resources: R.H.K. Visualization: D.O.A. Writing (original draft): D.O.A. Writing (review and editing): D.O.A., D.K.C., L.A.S., J.P.H., A.D.N., J.C.B., D.P., R.H.K., H.I.C., J.A.W., and M.D.S. Supervision: D.K.C., J.A.W., M.D.S., R.H.K., and H.I.C. Project administration: D.K.C. Funding acquisition (primary): D.K.C. Competing interests: D.K.C. is a co-founder of two University of Pennsylvania spin-out companies related to nervous system regeneration and restoration: INNERVACE Inc. and Axonova Medical LLC. J.P.H. is currently a paid consultant and an equity holder in INNERVACE Inc. There are two patent applications related to the methods, composition, and use of μTENNs. D.K.C. is an inventor on a U.S. patent application filed by the University of Pennsylvania (no. 15/032,677, filed 4 November 2013, published 1 September 2016) titled “Neuronal replacement and reestablishment of axonal connections.” D.K.C., J.P.H., J.A.W., H.I.C., and M.D.S. are inventors on a U.S. patent application filed by the University of Pennsylvania (no. 16/093,036, filed 14 April 2016, published 2 May 2019) titled “Implantable living electrodes and methods for use thereof.” The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

• Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

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Is 2021 finally the year for smart glasses? Here’s why some experts still say no

PUBLISHED SAT, JAN 23 202110:00 AM ESTSamantha Subin@SAMANTHA_SUBINSHAREShare Article via FacebookShare Article via TwitterShare Article via LinkedInShare Article via EmailKEY POINTS

• The tech industry has long worked to produce a fashionable, lightweight pair of consumer smart glasses that puts bulky headsets to shame, but experts say widespread adoption is five to ten years away.
• New designs are making headway in 2021, with Facebook, Vuzix and Lenovo models expected to hit the market as early as this summer, and an Apple offering possible by 2023.
• Smart glasses date back nearly a decade, but early consumer ventures like Google Glass deterred consumers with high price points and lack of linked content.

Facebook CEO Mark Zuckerberg delivers the keynote address at Facebook’s F8 Developer Conference on April 18, 2017 at McEnery Convention Center in San Jose, California.Getty Images

Big technology corporations including Apple, Alphabet’s Google and Microsoft have raced to create a pair of fashionable smart glasses readily embraced by consumers, but 2021 is shaping up to be one more year these gadgets fall short of mass adoption.

With a launch of Facebook smart glasses as early as this summer, technology companies are getting one step closer, but widespread consumer interest is five to 10 years away, says Tuong Nguyen, a principal analyst at technology consulting firm Gartner. And some tech CEOS agree. Snap CEO Evan Spiegel speculated it would be at least 10 years before consumers widely adopt smart glasses speaking during the 2019 TechCrunch Disrupt conference in San Francisco.

A recent report from Gartner forecasts the wearable device market will see significant growth, but that includes not only smart glasses but watches and virtual reality headsets. Gadgets like the Apple Watch already have been big successes. Gartner forecasts wearables will reach $109 billion by 2024, with watches and virtual reality headsets for gaming growing by compounded annual growth rates of 20% and 22%, respectively. ADVERTISING

For smart glasses, the lag in mass market adoption is a result of slow progress on easy-to-use designs, fashionable appearances and easy-to-use content, Nguyen said. Many products remain bulky, making it a hard sell for consumers looking to stay fashionable on the go, while slow connectivity issues dissuade those looking for efficiency. Companies also have yet to work out powering kinks such as whether its tethered to a computer or battery-powered.

But the latest iterations of smart glasses are showing progress. Last week, Vuzix premiered a fashion-forward product at the 2021 Consumer Electronics Show expected to hit the market this summer and that will feature speakers, noise-canceling microphones and offer options supporting WiFi and data.

New Vuzix smart glasses priced at $1,000 are expected to hit the market in mid-2021 and will mark the company’s first foray into the consumer end of what has been an enterprise market for this growing niche of wearables.Vuzix

The Rochester-headquartered company, which has long created smart glasses products for enterprise clients, is making its first major foray into the consumer market. In 2015, Intel Corp. invested nearly $25 million for a 30% stake in the company.

Lenovo introduced its ThinkReality A3 smart glasses at CES, which tether to a PC and some Motorola devices. These smart glasses allow users to project several virtual screens at once and come with an added speaker and microphone for interacting with peers or coworkers.

There’s also a range of everyday benefits to this product niche which continue to multiply as it develops. Smart glasses can be used for virtual shopping, figuring out what furniture fits into homes, and could one day replace screens entirely, said Rick Kowalski, director of industry analysis and business intelligence at the Consumer Technology Association.

For years, IKEA has toyed with using the Microsoft’s HoloLens to allow customers to plan out a kitchen, bathroom or room redesign, while Lowe’s implemented a pilot program in several stores in 2016. “The value that they bring is so far beyond what they can think of right now,” Kowalski said. 

A troublesome history

Smart glasses initially hit the market as early as 2012, but most have relied on enterprise clients, with only a few temporarily targeting consumers. 

In 2013, Google released the first prototype of the Google Glass to a select group of consumers before going public in May 2014. Retailing at $1,500 a pair, the product fit few consumer price points and came under fire for potential privacy issues related to its connected camera; it’s consumer model was discontinued in 2015, and the company found a place developing line for warehouse and manufacturing use.

The latest iteration geared toward the enterprise community was launched in 2019. Retailing at $999, the model comes equipped with voice activated applications can be managed from a mobile device.

Google Glass Enterprise Edition 2Google

Despite a slow integration into the consumer market, Covid-19 and the rise in remote work could accelerate smart glasses integration in health, medical and service fields. Vuzix recently announced they would partner with a medical technology company to assist with remote servicing during the pandemic.

Microsoft is also putting smart glasses to use in the defense sector. First available to consumers in 2019, the HoloLens glasses can overlay images and display computer programs. In 2019, the company won a $479 million contract to deploy 100,000 prototypes to the U.S. military hoping to improve soldier effectiveness, a move which came under harsh employee criticism.

The decision caused some concerns among employees who across the tech sectors have been more vocal in recent years as activists questioning some uses of their own technology. But Microsoft CEO Satya Nadella later defended the decision in an interview with CNN Business, citing that the company would not “withhold technology from institutions that we have elected in democracies to protect the freedoms we enjoy.”

In 2016, Snap made its foray into the smart glasses market with Spectacles, a wearable product that allowed users to record videos to sync with their phones. But by 2017, several media outlets reported that the creator of Snapchat largely overestimated demand and less than half of buyers continued using the product within a month of purchase, internal company data obtained by Business Insider suggested. Though largely unsuccessful among consumers, the most recent iteration offers 3-D capabilities and retails for $380, a fraction of the price of its competitors.

A person holds up a pair of Snapchat Spectacles by Snap Inc. on the floor of the New York Stock Exchange (NYSE) during the company’s initial public offering (IPO) in New York, March 2, 2017.Michael Nagle | Bloomberg | Getty Images

The price point for most smart glasses is still high, with many models upwards of $2,500. The new Vuzix glasses are expected to retail for $1,000; Lenovo has not yet detailed pricing on its ThinkReality A3 smart glasses. Offering consumer-friendly pricing for the products is one way to accelerate the rollout, but companies also need to ensure smart glasses are accompanied by easy-to-use apps and libraries of content that keep consumers occupied.

Without these vast content libraries released in tandem with smart-glasses, consumers are less likely to materialize the benefits of the product, said Allan Cook, a managing partner at Deloitte leading the digital reality practice.

“People think it’s weird to be wearing AR or smart glasses,” he said. “There’s been blocks to the market in the past, but in 2021 we are going to see some dramatic increase.”

Some experts thought 2020 would be “the year” of headsets due to the pandemic but it wasn’t, and again in 2021, while there will be growth it will be off a low base and more of it continuing in the enterprise market, according to a Deloitte analysis. Overall spending on AR and VR headsets, software, and services, including purchases by consumers, rose in 2020 to $12 billion globally, which was up 50% from 2019, but below the pre-pandemic forecast of almost 80% growth. With the pandemic accelerating the opportunity to demonstrate their value, digital reality headsets may continue to gain ground after the pandemic ends due to a variety of other benefits, such as lower cost, greater safety, and better learning retention, but those benefits still show more appeal to and adoption among enterprise clients rather than consumers, according to the Deloitte outlook.

Big Tech’s vision of the future

As technology companies push for greater consumer adoption, several tech giants beyond Google and Microsoft have announced plans for their own smart glass creations expected to hit the market in the next few years.

Facebook plans to release its smart glasses created with Luxottica’s Ray-Ban. The social media giant is banking on the popular sunglasses brand style-sense to offer a fashion-forward model that will appeal to skeptical customers. In 2019, CNBC also previously reported that the company was working on an accompanying artificial intelligence voice assistant to rival Amazon’s Alexa and Apple’s Siri.

“I can’t go into full product details yet, but they’re really the next step on the road to augmented reality glasses,” said CEO Mark Zuckerberg during a September livestream from Facebook Connect, where the company annually showcases virtual reality products.

Simultaneously, the company is working on Project Aria, an initiative that will distribute devices to employees to accumulate video, audio and location data, and ultimately help the company research and develop its smart glasses. Facebook first announced its Luxottica partnership in 2019.

Apple is reportedly working on its own AR glasses that would allow users to view maps, text messages and control Siri, expected to hit the market by 2023 at the earliest. Development on the product is shrouded in secrecy — like all of Apple’s R&D, which the company never provides comment on. An augmented and virtual reality headset similar to Facebook’s Oculus Rift is also reportedly in the works.

Wall Street is anxious for an Apple unveil, with Wedbush analyst Dan Ives putting Apple Glasses on his 2021 wish list and making a prediction that the company’s first augmented reality eyewear — the result of many years of development in the labs of Cupertino — will be introduced at the company’s WWDC 2021 developers conference, expected to be held in June.

Amazon has not announced plans for a full-scale augmented reality competitor, but it currently offers the Echo Frames, a pair of glasses enhanced with a speaker and built-in Amazon voice assistant.  

Jio, an India-based technology company that’s backed by both Google and Facebook, announced Jio Glass last year, a mixed reality headset that connects to the internet through a cable connected to a smartphone. Although the company, part of India’s largest conglomerate Reliance Industries, has yet to announce a price point, they’ve hinted that the product works with more than 25 applications and could benefit remote work and school.WATCH NOWVIDEO05:08Smart-glass startup View CEO says its business is poised for growth after it goes public via SPAC mergerRELATED

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What To Expect From Tesla In 2021

Jan 22, 2021 at 9:31am ET15+

By: EVANNEX

While 2020 was a hugely successful year for Tesla, 2021 stands to be even better.

This article comes to us courtesy of EVANNEX, which makes and sells aftermarket Tesla accessories. The opinions expressed therein are not necessarily our own at InsideEVs, nor have we been paid by EVANNEX to publish these articles. We find the company’s perspective as an aftermarket supplier of Tesla accessories interesting and are happy to share its content free of charge. Enjoy!

Posted on EVANNEX on January 22, 2021 by Denis Gurskiy

2020 was a significant year for Tesla. The Silicon Valley automaker absolutely rocked Wall Street in 2020 — winning Stock of the Year and minting ‘Teslanaires‘ young and old. Through Elon Musk‘s unique knack (or lack) of Public Relations, just about everyone learned about electric cars via some connection to Tesla last year. So what’s ahead for the EV leader in 2021?

Above: Cybertruck, ATV, Roadster, and Tesla Semi (Twitter: Elon Musk)

Well, this year is already off to a blistering pace. And there’s a lot to look forward to in Tesla Land this coming year. Here’s our preview of what to expect in the coming year. It doesn’t cover all the bases but it should provide a helpful overview for those who believe Tesla is just standing still. 

CYBERTRUCK

Okay, this is probably the most revolutionary (albeit controversial) vehicle coming through the Tesla pipeline in 2021. And there’ll be plenty of EV truck competition. That said, “We love this beast enough to have placed a pre-order.” Can’t wait. But, we’ll have to… hopefully 2021 will be The Year of the Truck for Tesla. Oh, and don’t forget — this year marks the arrival of Cybertruck‘s ultimate companion, the Tesla ATV.

TESLA SEMI

Speaking of trucks, there hasn’t been much news from Tesla on the Semi since it was first unveiled back in 2017. Musk has stated his desire to bring the Semi into volume production, and earnings reports state that 2021 is the new timeframe for that to happen. 

Despite delays, Tesla has continued to capture some large orders from a wide variety of companies for the Semi. Two of the more recent buys came from Walmart Canada for 130 trucks and Pride Group Enterprises for 150 trucks (with an option to increase to 500).

Will Tesla Semi launch in 2021? We’re excited to find out.

TESLA ROADSTER

Since Jay Leno got a well-publicized test drive, Tesla has also been somewhat secretive about its all-new Roadster. Heck, we didn’t even get to see those thrusters yet.

Unsurprisingly, with the pandemic, the Roadster was also pushed back. During one of the earnings calls this year, Musk stated that they’re hoping to start production of the Roadster sometime in mid to late-2021 with a possibility of being pushed back to 2022.

Additionally, some people who earned a discount on a Roadster through the Tesla Referral Program received an email from Tesla asking them to confirm whether they want to claim their discount or not. Perhaps the timing of these emails means it’s coming sooner than expected?

PLAID MODEL S

Rewind to 2019 — we witnessed a battle between the Tesla Model S and Porsche Taycan around the Nurburgring. In 2020, we did get some news regarding the ultra-high-performance Plaid Model S during battery day. The specs speak for themselves: $139,990 base price, 520+ mile range, 200 mph top speed, and under 2.0 second 0-60 mph time.

We do know that the tri-motor prototype Model S had an extra aerodynamic body kit on it during its Nurburgring runs, but we do not know precisely if the Plaid will bring any specific design changes to the interior (or exterior) of the car. Regardless, the Plaid Model S is to be expected sometime later this year.

GIGA SHANGHAI

In 2020, the Model 3 ramp at the factory has been impressive. Tesla states that the plant can now produce ~250,000 Model 3s moving forward. The Model Y portion of the factory is said to be able to produce ~250,000 Model Ys as well, bringing the total capacity of the Gigafactory 3 to (at least) half a million cars. Reports have also surfaced recently claiming Tesla should produce around 523,000 cars in 2021 at Giga Shanghai.

Could we see this kind of massive output in 2021? After all, that’s more vehicles out of one factory than Tesla produced worldwide in 2020. Only time will tell. But already we’re seeing Model Y deliveries in China this month.

GIGA BERLIN

With a proper foothold in China to address the Asian market, Tesla should be turning their attention to bringing vehicle production to the European continent. Giga Berlin is said to begin production later this year. The factory will be producing both the Model Y and Model 3 with a start date of sometime in mid to late 2021 as long as construction doesn’t see any major roadblocks. 

Interestingly enough, the Berlin Gigafactory could produce a Model Y that differs from ones made in Fremont and Shanghai. It looks like Berlin will be where Tesla tests some of its newer technologies like their new 4680 cells and/or upgraded paint system.

The Berlin Gigafactory could turn out to be the ultimate Tesla Gigafactory testbed site — where the company rolls out its newest features and tech. We’ll find out (hopefully) in 2021.

GIGA AUSTIN

Despite continuing work on both Giga Shanghai and Giga Berlin, Tesla’s new American Gigafactory resides in Austin, Texas. It should be up-and-running in 2021 in order to launch the company’s much-anticipated Cybertruck. Alongside the Cybertruck, Giga Austin will also produce the Model Y, because it seems like Americans can’t get enough of the Model Y. Giga Austin should evolve into an exciting story in 2021.

BETTER BATTERIES 

A better battery may be arriving in 2021. Tesla unveiled plans to vertically integrate battery production with their new 4680 battery cell. This new battery will allow Tesla to deliver more range and improved power for upcoming models. Not only that, but the design allows for a price reduction as well. While we have confirmation that these cells will be in the European Model Y, you can expect the 4680 cells to (ultimately) be available in the Semi, Roadster, and Cybertruck too.

Tesla’s pilot plant is expected to be completed sometime in 2021 to kick off the next chapter for Tesla’s batteries. We recently got a preview of what the production line might look like as it rolls out soon.

AND MORE…

What about a completely redesigned version of the Model S and Model X. Will there be more updates to Tesla’s solar business and solar roof options? Will more utilities take advantage of Tesla’s energy storage and Autobidder capabilities? What about improvements to Autopilot? And what can we expect from future software updates?

There’s a lot more to expect from Tesla in 2021. For a deeper dive into the topic of Tesla’s 2021 forecast (especially as it relates to the stock), check out the video below. In addition, we’re sure, there are things yet-to-be-mentioned that will come across Elon Musk’s Twitter feed. We’ll be watching.

VIDEO

Above: Rob Maurer and Sean Mitchell look ahead at what investors should be thinking about as it relates to Tesla in 2021 (YouTube: Sean Mitchell)

15 Genius Morning Routine Tips For Night Owls

#1: Break up with your snooze button.

Rawpixel.com/ShutterstockBy Kathleen FerraroJan. 22, 2021

If you thrive in the darkness, chances are you’re not going to become a morning person overnight. In fact, science proves that some people naturally feel more productive in the evening.That doesn’t have to mean you can’t improve your a.m. habits. To help, experts are sharing their morning routine for night owl tips that’ll make waking up less of a drag.

“People who have energy at night can have a hard time finding that same energy in the morning,” says Nawal Alomari, LPC, a licensed counselor and life coach. “When you wake up in a rush or with stress because of a late night, the whole day feels like you’re trying to catch up.” That’s where having a morning routine comes in to help: According to Alomari, the first part of your a.m. can really set the tone of your day. “Taking time for yourself can make you feel more relaxed and proactive,” she says.

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Sure, going for daily sunrise runs, making pancakes, and doing a meditation all before 8 a.m. may be a pipe dream for night people, but fear not: here are 15 morning routines for night owls that’ll help you slay the first part of your day without the need for a super-early wake time.

1. Be Consistent

Despite your best attempts, night owls often can’t help but stay up late. And that’s OK, says wellness coach Erin Clifford, JD, as long as you have a consistent sleep schedule (and yes, that includes weekends). “Whether you’re a night owl that sleeps in or has to wake up early, keep those times consistent,” she tells Bustle. “When we regularly go to sleep and get up at the same time, then our bodies get used to that routine.”

Besides helping you rise and shine, research shows that regular sleep helps you think clearly, focus, feel good, and even reduces your risk for heart disease, mood disorders, and other illness, which means there are plenty of reasons to have a nightly bedtime.

2. Resist The Urge To Snooze

It’s time to acknowledge that the snooze button is your frenemy, and that it’s time to end this toxic relationship. “You want to get up when the first alarm goes off because the sleep you get after that is not good sleep,” says Debra Swan, a certified health coach and personal trainer. Those extra 10 minutes of ZZZs actually hurt more than they help: One study found that disrupting your sleep, which can happen when you blow through five morning alarms, ramps up stress, messes with your ability to think clearly, and can even put you at higher risk for disease (such as cardiovascular disease).

Her tip for kicking the habit? “Put the alarm out of arm’s reach so you have to get out of bed to turn it off,” says Swan. “If it’s hard, try doing it in increments by hitting snooze six times one day, then only five times the next.”

3. Create Your Ideal Morning Routine, Then Make It Mini

Ambitious morning plans may sound like a good idea the night before, but when the time comes to wake up, it’s easier said than done. That’s why Alomari suggests mapping out your dream morning routine, then shrinking it down to just 10 or 20 minutes. “In my ideal world, I’d wake up at 6 a.m., drink some water, make my bed, work out, have some breakfast, and start my job,” she says. “Instead, minimize that routine to the best or most important parts. If you enjoyed the 10 minutes you set aside for a mini routine, that positive reinforcement will make you want to keep doing it.”

The idea is that over time, you’ll be motivated to expand it. Consider it a highlight reel of the morning routine you could one day do on the daily.

4. Soak Up The Sun

Catching some rays first thing in the a.m. can help a night owl adjust to daytime, according to Swan. “Get bright, natural light first thing in the morning,” she says. “Open your curtains and get a face full of sunlight if possible, because this will synchronize your circadian rhythm and tell your brain that it’s time to be awake.” As an added bonus, vitamin D from sun exposure can also help promote immunity, strengthen your bones, and boost your mood.

For those who are stuck in grayer locations or who rise before the sun, you can trick your body into daylight mode by turning on all your bedroom lights in the meantime, says Swan.

5. Hydrate, Hydrate, Hydrate

If it feels like water is the answer to everything, that’s because it usually is. Keep a large glass of water next to your bed so you can gulp it down first thing in the morning, says Swan.

“Lots of people grab a big cup of coffee first thing in the morning,” she tells Bustle. “But you want to have water before that.” That’s because it rehydrates your body after a night’s sleep, boosts your metabolism, and helps you feel energized. Plus, coffee can be dehydrating and sometimes irritate an empty stomach, so go for the water first. “No one feels energized when they’re dehydrated,” says Swan.

6. Keep A Caffeine Journal

Caffeine is the elixir of many a night owl. But even though it might help you wake up (after that water, of course), ingesting caffeine too late in the day can mess with your sleep and set you up for a tough time the next morning, according to Clifford. She suggests tracking how your favorite brew affects your energy so you can figure out the best caffeine cut-off time for you.

“Caffeine affects everybody differently,” says Clifford. “You might be fine drinking coffee until 2 p.m., but that might not work for others. Keep a journal of when you have caffeine and alcohol, even for just a week or two, to see how it affects your energy levels and sleep.”

7. Separate Your Morning Routine From Your Workspace

If you’re working from home, the lines between work and personal time are blurred. That’s why Alomari recommends doing your morning routine in a different area from your work station if possible. “Starting your morning in a different room, like in your kitchen with a cup of tea or outside on your patio, separates your downtime from the place where your responsibilities lay,” she tells Bustle. This can help create some much-needed boundaries to help you get the most out of your me time.

8. Have Something To Look Forward To

It may sound obvious, but if you hate mornings, try to make them less hate-able. By starting your day with an activity you enjoy, then you’re more likely to feel positive about your morning, according to Swan (yes, it’s possible).

“If you’re going to dread the things you have to do in the morning, like cleaning up last night’s dishes, you’re not going to look forward to waking up,” she tells Bustle. “Build a routine that incorporates things you like. Do your favorite yoga video, walk with a friend, listen to a podcast, or try a new waffle recipe. If you’re excited to do something specific in the morning, you’re more likely to hop out of bed.”

9. Eat Some Protein

There’s some truth to the old “breakfast is the most important meal of the day” cliche. Clifford says fueling with protein-rich snacks like eggs, yogurt, or nuts can give you a leg up on energy throughout the day. If you’re crunched for time, Swan recommends overnight oats as an easy on-the-go breakfast.

And there’s research to back it up — one study found that not only can eating protein in the a.m. help you maintain energy levels all day long, but it can boost your longterm health.

10. Try A Morning Workout

If waking up doesn’t come easy, start your day with activity to turn on your body and mind, suggests Swan. If you’re able to knock out a full workout in the morning, all power to you, but a walk outside or a few minutes of simple bodyweight exercises will also do the trick, she says.

Morning activity can also help you feel your best throughout the day. “When you exercise, it releases endorphins that can put you in a good mood, help you feel energized, and make you feel sleepy at night so you get more or better rest,” says Swan.

11. Make a List

List makers, it’s your time to shine. Oludara Adeeyo, ASW, a psychiatric social worker and psychotherapist, suggests starting your morning by writing a list of what you’d like to accomplish in the day — this helps you map out your time and set expectations. “Lists help guide your focus for the day,” she tells Bustle. “You can prioritize what items are most important and tackle them accordingly.” It can also help you relieve anxiety, improve your memory, and feel good about what you’ve accomplished.

12. Set The Thermostat

Nothing will force you back under your covers faster than a freezing bedroom. Enlist your smart thermostat in the battle against rough mornings, recommends Swan. Program your thermostat to go down to about 65°F at night and then rise back up in the morning so that you can roll out of bed in comfort, she says.

13. Plan Ahead

Remember how your parents made you pick out your school clothes the night before? Turns out they were onto something. Whether it’s laying out athletic wear for your morning workout or prepping your breakfast before bed, getting a jump on logistics can save you time for extra sleep or more enjoyable morning activities.

“I swear by this,” says Adeeyo. “This will improve productivity because you can focus your morning time on simply getting ready for the day.”

14. The 20/20/20 Rule

Alomari recommends the 20/20/20 trick to prep your brain for restorative sleep, which can help you the next morning. Start by spending 20 minutes (or 10 minutes, 30 minutes, or an hour) doing whatever you normally do before bed, like watching TV or playing on your phone. Then spend 20 minutes doing something without a screen, like reading or journaling. After that, lie down for 20 minutes to unwind.

“This lets your brain process the day, and your brain will tire itself out,” says Alomari. “That whole time, you’re prepping yourself to fall into a healthy sleep.”

15. Tackle Your Anxiety

Some people who stay up late aren’t doing it by choice: Both stress and anxiety can cause you to toss and turn long after your bedtime. Science shows that having an anxiety disorder can contribute to sleep disturbances or insomnia. “Make sure the reason you’re up at night isn’t negative, because if it is, a morning routine won’t fix that,” says Alomari. “Someone who’s up late with anxiety usually wakes up with anxiety.”

If that sounds like you, Alomari recommends speaking to a therapist to address the underlying cause of your anxiety and help your body return to its natural sleep rhythm.

Studies referenced:

Clark, I. (2017). Coffee, caffeine, and sleep: A systematic review of epidemiological studies and randomized controlled trials. Sleep Med Rev. https://pubmed.ncbi.nlm.nih.gov/26899133/

Kamada, I. (2011). The impact of breakfast in metabolic and digestive health. Gastroenterology and Hepatology from Bed to Bench, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4017414/

Medic, G. (2017). Short- and long-term health consequences of sleep disruption. Nature and Science of Sleep, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449130/

Philpott, J. (2020). Casein kinase 1 dynamics underlie substrate selectivity and the PER2 circadian phosphoswitch. eLife, https://pubmed.ncbi.nlm.nih.gov/32043967/

Staner, L. (2003). Sleep and anxiety disorders. Dialogues in Clinical Neuroscience, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3181635/

Worley, S. (2018). The Extraordinary Importance of Sleep: The Detrimental Effects of Inadequate Sleep on Health and Public Safety Drive an Explosion of Sleep Research. Pharmacy and Therapeutics, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281147/

Experts:

Oludara Adeeyo, A.S.W., a psychiatric social worker and psychotherapist in Los Angeles

Nawal Alomari, L.P.C., a licensed clinical professional counselor and life coach based in Chicago

Erin Clifford, J.D., a certified holistic wellness coach in Chicago

Debra Swan, a certified health coach and personal trainer based in Chicago

20 Ways to Sleep Better Tonight

CR offers advice on revising your routines and tweaking your bedroom to get the shut-eye you crave—and need

By Haniya Rae, with additional reporting by Lisa LombardiJanuary 22, 202121 SHARESILLUSTRATION: JOEL HOLLAND

Sleep is critical to our health. But for many of us, a restorative night’s slumber is another casualty of the COVID-19 pandemic at the very time we all need it the most. In a nationally representative CR survey (PDF) of 2,851 U.S. adults last November, 28 percent of Americans reported having more trouble falling or staying asleep since the pandemic hit the U.S.

“Not getting enough sleep can weaken the immune system,” says Rafael Pelayo, MD, a sleep specialist and clinical professor of psychiatry and behavioral sciences at Stanford University and author of “How to Sleep: The New Science-Based Solutions for Sleeping Through the Night” (Artisan 2020). “If you add on top of chronic sleep deprivation the stress of an infection, our system can be overwhelmed.”

Poor or insufficient sleep can dull your brain and has been linked to an increased risk of serious illnesses such as cancer and cardiovascular disease, according to the National Institute of Neurological Disorders. The National Institutes of Health has found that improving sleep can help protect against COVID-19, making it more important than ever.

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Using our bedrooms as work and exercise spaces or even as classrooms, as many people are doing now, only makes getting a good night’s sleep more difficult, experts say.MORE ON SLEEPCR’s Mattress Ratings & Buying GuideBest Mattress Cooling Pads From Consumer Reports’ TestsBest Mattresses of 2021CR’s Sheet Ratings and Buying GuideCR’s Pillow Ratings and Buying Guide

“We typically tell patients that the bedroom is only for two things: sleeping and sex,” says Mathias Basner, MD, a professor in the division of sleep and chronobiology at the University of Pennsylvania. He explains that limiting the bedroom to those activities sends a clear signal to your brain when it’s time to hit the sack. “But obviously we realize that not everybody may have that option.”

No matter how much tossing and turning you’re doing these days, we’re here to help you get on track to better sleep. We explain the smartest ways to prepare for solid snooze time and steer you toward the mattress, pillows, and sheets that will keep you cozy all night long. For those who wake to find themselves baking under the covers at night, we offer a review of mattress cooling pads. And if you’re among the many using a bedroom for double duty, we have tips to minimize the mixed message this sends to your brain. We even offer expert advice on ways to start your day that can lead to a better night’s sleep. (To skip to a section, click on the links below.)

How to Get Ready for Bed

Make a Bed You Can’t Resist

Power Up in the A.M.

The Double-Duty Bedroom

How to Get Ready for Bed

ILLUSTRATION: JOEL HOLLAND

A healthy bedtime routine can help you make the transition from keyed-up to Jell-O-limbed and prepare your body and mind to melt into slumber’s warm embrace. The key is to ease into it. Follow these tips.

1. Try Meditation
There’s some evidence that meditating during the day can improve sleep at night. A study published by the Journal of the American Medical Association found that mindfulness meditation improved sleep in older adults with sleep disturbances. Resources are widely available online, including from the Center for Mindfulness at the University of California at San Diego. Some research shows that widely available apps such as Calm and Headspace, which use guided imagery and even bedtime stories to encourage sleep, are effective.

2. Give Your Belly a Break
Stuffing yourself can make you groggy, but experts say that going to bed with a full stomach can cause reflux, which can wake you up. Sue X. Ming, MD, a professor in the neurology department at the Rutgers New Jersey Medical School, recommends staying away from heavy meals 3 to 4 hours before bedtime. “Especially avoid foods with a high fat content or dense carbohydrates, which stay in the stomach longer,” she says.

3. Put the Plug in the Jug
If sales figures are any indication, people are drinking more during the pandemic. Sales of spirits and hard liquor increased by 33 percent during the first six months of 2020 compared with all of 2019, according to Ibotta, a cash-back app that rewards people for everyday purchases. A drink or two might seem like a good way to quiet the mind before bed, but experts say that alcohol actually decreases rapid eye movement (REM) sleep, a critical phase of the sleep cycle that helps our emotional equilibrium and enables us to retain things we learn during the day. “Alcohol will get you to sleep,” Ming says, “but you’ll wake up in the middle of the night.”

4. Turn Down the Temp
The National Sleep Foundation recommends keeping bedrooms at a cool 65° F for optimal sleep. Being too hot or losing too much heat can interrupt REM sleep and cause people to wake up, Ming says. A programmable thermostat can be set to cool a room just right in the wee hours. Another way to regulate your body temperature is to use a mattress cooling device.

5. Ease Into Bedtime
Building wind-down time into your routine is a good way to prepare your mind and body for sleep. Sara Benjamin, MD, a clinical associate and instructor in neurology at the Johns Hopkins Center for Sleep, recommends a personal-care ritual (washing your face, brushing your teeth, etc.) an hour before bedtime, and then moving on to relaxing activities until it’s lights out. These could include reading, listening to music, writing in a journal, praying, meditating, or doing gentle forms of yoga such as restorative, yin, or yoga nidra, all of which involve little or no movement. Just be sure that whatever you do is calming: no rock and roll, horror flicks, or murder serials.

6. Turn Off the TV (and All Other Screens)
Bright light and the blue light produced by TVs, cell phones, and computers can suppress the production of melatonin, the hormone responsible for regulating sleep. “Cell phone displays are extending the day into the night,” says Lisa Ostrin, OD, an associate professor at the University of Houston College of Optometry. She says that the nighttime mode on devices may reduce the effect of blue light somewhat, but not the stimulating effect that reading email can have on your brain. Sleep experts recommend turning off all screens and dimming bedroom lights at least 30 minutes before bedtime.

7. Fade to Black
Heavy curtains or blinds will reduce light exposure and can improve sleep. “Anything that blocks out light helps you stay asleep or sleep more soundly,” says Ming, adding that darkness promotes the secretion of melatonin, too.

8. Take Some Sound Advice
“If environmental noises disturb your sleep, white noise can be very helpful,” Benjamin says. Generated by a white-noise machine or certain apps, white noise masks sounds—such as honking horns or slammed doors—that wake you or keep you from falling asleep.

9. Consider a Sleep Tracker
Devices such as the Oura Ring are becoming increasingly popular and more sophisticated. (Some fitness trackers and smart watches also offer sleep tracking capabilities.) Research has found that they can do a good job of tracking the overall time you sleep and encouraging good habits by, for instance, reminding you when to power off devices and begin getting ready for bed. Some can even track environmental factors, such as the light and temperature in a bedroom. Benjamin notes, however, that they can’t accurately track sleep stages. Even so, some people find the data on sleep time, as well as on heart and respiration rates and body temperature, useful for gauging how factors like exercise and alcohol may affect their sleep. In rare cases, people can become obsessed with their sleep stats and lie awake at night worrying about what the data will reveal in the morning. If you’re someone who really digs data on your sleep habits, go for it. But if the device becomes one more thing you worry about, it’s better to ditch it.

Make a Bed You Can’t Resist

Follow these tips to create a bed you can’t wait to get into at night.

10. Assess Your Mattress
One that isn’t supportive and doesn’t properly distribute your body weight can cause joint or muscle pain, make you restless, and interfere with your sleep. If you feel sore in the morning or if your mattress feels lumpy, has a permanent depression in it, or is more than 10 years old, it’s probably time for a new one. In our tests, we rate mattresses for how well they support various body types. We also point you to the brands that are the most comfortable based on feedback from 73,676 CR members who purchased one within the past decade. See CR’s ratings of innerspring, foam, and adjustable air mattresses.

11. Pick a Perfect Pillow
The more a pillow keeps your neck and spine naturally aligned, the more comfortably you’ll sleep. “Try to find a pillow that keeps your neck in as neutral a position as possible and doesn’t crane it in any position,” says Joel Press, MD, physiatrist in chief at the Hospital for Special Surgery in New York City. A supportive pillow can prevent excessive motion and keep your neck in a better position without straining it. The Coop Home Goods pillow, for $60, tops our ratings. You can customize its support by adding or removing filling, and it comes with a generous 100-night sleep trial policy.

12. Choose Your Sheets Wisely
As with all bedding, the most important thing about sheets is that they’re comfortable. To rate cotton sheets, we test how well they fit on a mattress after a year’s worth of washing, how much they wrinkle, and how easily they tear. Our testing panel also grades them for softness. L.L.Bean’s Pima Cotton Percale sheets are nearly as good as the top-ranked Matouk Sierra but are far less expensive ($149 for a queen set vs. $258 for a single fitted sheet). The L.L.Bean set shrank only slightly after a year’s worth of washing, and like the Matouk was judged to be soft. Pelayo says that sleeping on clean sheets can lessen allergic reactions to dust mites, which can make you congested and agitated. CR recommends washing sheets at least once every two weeks.

13. Try a Weighted Blanket
Long used to calm children with autism or behavioral disorders, weighted blankets have caught on with the general public as a way to improve sleep. Sales of these heavy, quilted bed coverings (some weigh up to 35 pounds) were soaring even before the pandemic. “The idea is that weighted blankets give a sense of comfort and may facilitate the secretion of oxytocin,” Pelayo explains. “But to what degree and whether it’s a short-term or long-term effect is not clear. I have patients who like weighted blankets and others who don’t. Those who like them really like them.”

The rule of thumb for choosing a weighted blanket is that it should be about 10 percent of your body weight. Popular models include the YnM (starting at $70) and the Harkla (starting at $110) for a 15-pound blanket. Both are available in weights up to 25 pounds.

Power Up in the A.M.

No matter which side of the bed you wake up on, the way you manage your mornings can set the tone for the entire day. “Our wake-up routine is just as important to the day ahead as a relaxing nighttime routine is to sleep,” says Kien Vuu, MD, an assistant professor of health sciences at UCLA and author of “Thrive State: Your Blueprint for Optimal Health, Longevity, and Peak Performance” (Lifestyle Entrepreneurs Press, 2021).

Here, the habits that will set you on course to a less stressful, more productive day.

14. Get a Head Start
The right way to start your day actually begins the day before, according to Alexis Haselberger, a productivity coach in San Francisco. She suggests devoting your last 15 minutes at work to making a to-do list for the following day. This allows you to mentally disconnect from work and avoid starting the next day in a chaotic state of mind wondering what to do first.

15. Let There Be Light
A small study published in the journal Behavioural Brain Research found that sleep-deprived people exposed to simulated, slowly increasing sunlight in the morning did significantly better at attention-based tasks throughout the day than those exposed to steady dim light. Based on that finding, you might consider using an alarm clock that wakes you by gradually bathing your bedroom in light. Once you’re awake, soak up some sunshine ASAP. “Getting 10 to 20 minutes of direct sunlight just as soon as we’re out of bed resets our circadian rhythms,” Vuu says, “making it easier to fall asleep at bedtime.”

16. Take a Tech Break
Resist the temptation to check your email, texts, Slack channels, or other electronic communication first thing in the morning or even at the start of your workday. Haselberger says that reserving the first hour of your workday for other tasks helps give you a sense of control over your day. “It rarely makes a difference to others whether we respond to messages at 9 or 10 a.m., but you can get an incredible amount done in that hour if you avoid checking email,” she says. “[Those] are other people’s priorities, but because we are social creatures, the second we’ve read a message, we feel compelled to respond.”

Depending on your profession and how demanding your boss is, your “off the grid” time may be more like 10 to 15 minutes, but that’s fine, too.

17. Begin the Day by Completing a Task
Quickly making your bed while the coffee brews or putting away the dishes while you wait for your toast can give you a feeling of accomplishment right off the bat. “It’s easier to stay productive if you’ve started the day with a small accomplishment,” Haselberger says.

The Double-Duty Bedroom

ILLUSTRATION: JOEL HOLLAND

While experts agree that bedrooms should ideally be reserved solely for sleeping and sex, reality is often far from that ideal. In our survey (PDF), 45 percent of Americans told CR they’ve regularly used their bedroom for at least one other purpose since the pandemic began. (More than 7 in 10 of them said they were doing so even before the pandemic.) Since the pandemic began, though, Americans say they have started using their bedrooms for purposes they didn’t before: as a home office (11 percent), dining room (6 percent), hobby space (6 percent), exercise room (6 percent), meditation space (6 percent), or classroom (5 percent). If you’re among them, the following tips will help limit any negative impact this may be having on how you sleep.

18. Reduce Work Reminders
If you need to use your bedroom as a workspace, experts recommend taking an “out of sight, out of mind” approach. “Clutter, work papers, even phones and other connected devices signal to the brain and body that there is work to be done or a problem to be solved, making it harder to sleep,” says Janet Kennedy, PhD, a psychologist and founder of NYC Sleep Doctor. Effective ways to minimize reminders of work include using a laptop rather than a desktop so you can stow it away at the end of the day, tucking your desk into a corner of the bedroom, or using a bedside table for your desk during the day and restoring it to its original purpose when it’s time to clock out. The same advice goes for bedrooms that double as classrooms during the day.

19. Create an Exercise Zone
Exercise, like sleep, is vital to good health. But having hand weights, yoga blocks, and other exercise equipment lying around a bedroom can “trigger rumination and increased brain activity, which can cause insomnia and erode sleep quality,” Kennedy says. So stash them in your closet or put them in a storage container under the bed when you’re not using them. Joanna Teplin, co-founder with Clea Shearer of The Home Edit, a full-service home organization company, recommends thinking of your bedroom the way someone with a studio apartment might, giving each area its own purpose. Place large equipment like treadmills as out of the way as possible, and consider using decorative screens or room dividers to hide them from view.

20. Control Kids’ Clutter
Their art projects and toys have a way of migrating to the primary bedroom. Create a pristine sleep environment by putting everything in bins and placing them in another area of the home at the end of the day, Shearer says.

Editor’s Note: This article also appeared in the March 2021 issue of Consumer Reports magazine.

PORTING FIREFOX TO APPLE SILICON: TALES FROM THE TRENCHES

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January 22, 2021

For any smaller and larger software product that aims to be compatible with Apple’s MacOS, the recent introduction of its ARM-based Apple Silicon processors and MacBooks to go with them came as a bit of a shock. Suddenly one of the major desktop platforms was going to shift processor architectures, and with it likely abandon and change a number of APIs. Over at Mozilla HQ, they assumed that based on past experiences, Apple’s announcement of ‘first Apple Silicon hardware’ would also mean that those systems would be available for sale.

Indeed, one week after the November 10th announcement Apple did in fact do so. By then, Mozilla had worked to ensure that the Firefox codebase could be built for Apple Silicon-based MacOS. Fortunately, through the experiences of running Firefox on Windows-on-ARM, they already had gained a codebase that was compatible with 64-bit ARM. Ultimately, the biggest snag here was the immature Rust language and dependency support for Apple Silicon, which set back the first release.

When it came to the distributing of Firefox on Intel- and ARM-based Macs, the decision was made to package both versions of the application into a so-called Universal Binary. While this pads out the size of the installer, it also means easier distribution and would not affect the built-in updater in Firefox. This also allowed for an easy fix for the Google Widevine DRM module, for which no Apple Silicon version was available at first, allowing the same module for Intel to be used with either Firefox version via the Rosetta 2 binary translator in MacOS (as we covered previously).

After this it was more or less smooth sailing, with some Rosetta 2-based glitches and MacOS Big Sur-related bugs that spoiled some of the fun. What this experience shows is that porting even a big codebase like Firefox to Apple’s new platform is fairly straightforward, with lack of support from toolchains and other dependencies the most likely things that may trip one up.

The Rosetta 2 feature, while helpful, also comes with its share of gotchas as the Firefox developers found out, and of course there is a lot more optimization that can (and should) be done for such a new platform.Posted in News, Software DevelopmentTagged Apple Silicon, arm, firefox